THE HOST-PARAS ITE RELATIONSHIP BETWEEN NIGRESCENSsummit.sfu.ca/system/files/iritems1/4295/b1363012x.pdf · 2020-03-30 · , the desert locust, has been an important crop pest for

THE HOST-PARAS I T E RELATIONSHIP BETWEEN MERMIS NIGRESCENS

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DUJARDIN AND THE DESERT LOCUST, SCHISTOCERCA GREGARIA F'ORSKAL .

STUART MELVYN CRAIG

B. Sc. , S i m o n Fraser U n i v e r s i t y , 1970

A T H E S I S SUBMITTED I N PARTIAL FULFILLMENT O F

THE REQ-u-IREMENTS; THE; jjEGKEE O F

MASTER O F S C I E N C E

i n t he d e p a r t m e n t

of

~ i o l o g i c a l Sciences

@ STUART M E L W N CRAIG 1 9 7 3

SIMON FRASER UNIVERSITY

A p r i l , 1 9 7 3

~ r o n t i s p i e c e

Adult d e s e r t l o c u s t i n f e c t e d wi th Mermis n i s rescens

APPROVAL

Name : S t u a r t Melvyn Craig

Degree : Master of Science

T i t l e of Thesis : The h o s t - p a r a s i t e r e l a t i o n s h i p between Mermis n iq rescens Dujardin and t h e d e s e r t l o c u s t , Sch i s toce rca g r e q a r i a ~ o r s k s l

Examining Committee:

Chairman: R.M.F .S. Sadleir

J. M. Webster . - Senior Supervisor

J. H. Borden

Thelma ~ i n l a ~ s o n

Date Approved:.((

P A R T I A L COP'tRIGHT L I C E N S E

I hereby g r a n t t o Simon F rase r Univers i ty t he r i g h t t o lend

my t h e s i s o r d i s s e r t a t i o n ( the t i t l e of which is shown below) t o u s e r s

of t he Simon F rase r Univers i ty L ib ra ry , and t o make p a r t i a l o r s i n g l e

copies only fo r such u s e r s or i n response t o a reques t from the l i b r a r y

of any o the r u n i v e r s i t y , or o the r educa t iona l i n s t i t u t i o n , on i t s own

behal f o r f o r one of i t s u s e r s . I f u r t h e r agree t h a t permission f o r

mu l t ip l e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted only

by me X K X ~ ~ X & P ~ # ~ Z D R ~ ~ ~ . It is understood t h a t copying

o r publ ica t ion of t h i s t h e s i e f o r f i n a n c i a l g a i n s h a l l no t be allowed

without my w r i t ten permission.

T i t l e of Thesis / ~ i s s e r t a t i o n :

The Host-Parasite Relationship between M e r m i _ _ . s w ~ . w e n s

~ujardin and the Desert Locust, Schistocerca ~"eqaria Forska:

Author : - ( s i g n a t u r e )

Stuart Melvyn Craia

(name)

A p r i l 16th, 1973.

(da te )

ABSTRACT

There a r e s e v e r a l important a spec t s of t h e hos t -

p a r a s i t e a s s o c i a t i o n between Mermis niqrescens and t h e d e s e r t

l o c u s t , Schis tocerca q r e q a r i a , which must be i n v e s t i g a t e d

before t h i s nematode can be c u l t i v a t e d i n vivo f o r use i n the -- b i o l o g i c a l c o n t r o l of t h e d e s e r t l o c u s t . The purpose of t h i s

i n v e s t i g a t i o n was t o s tudy the development of g. niqrescens

i n S. q r e q a r i a i n t h e l abora to ry , t o determine t h e i n t e r a c t i o n

between - M. n iqrescens and 2. g r e q a r i a , and t o determine t h e

mechanisms of M. - niqrescens pa thogen ic i ty on 2. q r e q a r i a .

n iqrescens eggs hatched w i t h i n 3 1/2 hours

a f t e r they were fed t o 5. q r e q a r i a a d u l t s , i n t h e region of the . h o s t g u t between t h e p o s t e r i o r end of t h e crop and t h e p o i n t

of o r i g i n of t h e Malpighian tubu les . The nematode penet ra ted

i n t o the haemocoel i n t h e same reg ion a s egg ha tching 'occurred ,

bu t 1 t o 2 0 hours l a t e r . P a r a s i t e eggs remained v i a b l e f o r

up t o 27 months i n d ry s t o r a g e a t 5 ' ~ and 55-60% R.H.

Severa l f a c t o r s inf luenced t h e dura t ion of t h e

p a r a s i t i c phase and t h e u l t ima te s i z e of t h e nematode i n the

l o c u s t . A s t h e number of p a r a s i t e s increased the t o t a l

development time shortened. I n f e c t e d l o c u s t s reared a t 3 5 ' ~

shortened t h e t o t a l p a r a s i t e development time and s i g n i f i c a n t l y

iii

lengthened t h e nematodes as compared w i t h t h o s e r e a r e d a t

2 2 ' ~ . P a r a s i t e s remained f o r a s i g n i f i c a n t l y l onge r p e r i o d

of t ime and grew s ign i -T ican t ly more i n l e n g t h i n female h o s t s

t h a n i n male h o s t s . They remained f o r a s i g n i f i c a n t l y l onge r

t i m e i n o l d e r a d u l t h o s t s t h a n i n ones t h a t had j u s t completed

t h e imaginal moul t . Larvae deve lop ing from eggs f e d t o a d u l t

l o c u s t s had a f a s t e r growth r a t e and grew s i g n i f i c a n t l y more

i n l e n g t h than t h o s e deve lop ing from eggs fed t o nymphal

h o s t s . The d u r a t i o n o f t h e p a r a s i t i c phase was dependent on

t h e n u t r i t i o n a l s t a t e o f t h e h o s t . Nematodes i n c r e a s e d

s i g n i f i c a n t l y i n l e n g t h and remained a s i g n i f i c a n t l y l onge r

p e r i o d o f t ime i n two c o n s e c u t i v e h o s t s t han d i d nematodes

t h a t had developed solely in one host,

The sex r a t i o o f t h e deve lop ing nematode l a r v a e w a s

i n f luenced by t h e number o f nematode p a r a s i t e s i n t h e h o s t ,

and by t h e weight o r age o f t h e h o s t . I n c r e a s e i n t h e

i n f e c t i v e burden ( i . e . , t h e number of nematodes) r e s u l t e d i n

more males , b u t t h e minimum i n f e c t i v e dose r e q u i r e d t o cause

male nematodes t o develop was independent o f h o s t weight below

a c e r t a i n dose l e v e l , and s t r o n g l y dependent on h o s t weight

above t h a t l e v e l w i t h i n each h o s t age c l a s s .

Depending on h o s t age a t i n f e c t i o n , t h e r e w a s a

i v

c e r t a i n minimal i n f e c t i v e dose above which t h e next-but-one

h o s t moult was i n h i b i t e d . The o l d e r t h e h o s t a t i n f e c t i o n ,

t h e h i g h e r t h e burden o f p a r a s i t e s r e q u i r e d t o i n h i b i t

moul t ing. M. n i q r e s c e n s was demonstra ted t o i n h i b i t h o s t

moul t ing by s i g n i f i c a n t l y d e c r e a s i n g t h e ra te o f f a t body pro-

t e i n s y n t h e s i s , by d e c r e a s i n g t h e ra te o f amino a c i d

p r e c u r s o r i n c o r p o r a t i o n from t h e haemolymph and c o n c u r r e n t l y ,

by p o s s i b l y s t i m u l a t i n g ca t abo l i sm o f f a t body p r o t e i n s t o

p rov ide a d i e t a r y sou rce o f amino n i t r o g e n f o r i t s own

development.

ACKNOWLEDGEMENTS

I wish t o express my g r a t i t u d e t o D r . J. M. Webster

f o r h i s u n f a i l i n g guidance, indomitable enthusiasm and en-

dur ing pa t i ence throughout t h e p repara t ion and w r i t i n g of

t h i s t h e s i s . I am a l s o indebted t o t h e o the r members of my

Supervisory Committee, D r s . P . Belton, J. H. Borden and

K. K. Nair f o r t h e i r h e l p f u l comments and sugges t ions through-

o u t my resea rch . I wish t o e s p e c i a l l y thank D r . R. Gordon

(Department of Biology, Memorial Univers i ty of Newfoundland,

S t . ~ o h n ' s ) f o r h i s a s s i s t a n c e dur ing the e a r l y p a r t of my r e -

sea rch and h i s h e l p f u l comments dur ing t h e f i n a l p repara t ion

~f *is manuscr ip t , P rz fecsz r 9 . e l x . B i n l a y s ~ n fcr her helpful

sugges t ions , Mr. W. Dean f o r h i s valuable t e c h n i c a l a s s i s t a n c e

i n r e a r i n g S. g r e q a r i a , D r . P. T. Haskel l (Centre f o r Overseas

Pes t Research, London, ~ n g l a n d ) f o r h i s u s e f u l l i t e r a t u r e on

S . q r e q a r i a , D r . D. S . King (Zoecon Corporat ion, Palo Al to , -

C a l i f o r n i a ) f o r h i s advice on t h e b ioassay f o r ecdysone,

M r . R. Long f o r h i s p r o f e s s i o n a l advice and a s s i s t a n c e i n photo-

graphy, and Pamela Timms f o r typing t h e t h e s i s . F i n a l l y , I

express my a p p r e c i a t i o n t o D r . G. H. Geen and the Department

f o r providing t h e f a c i l i t i e s and t h e oppor tuni ty t o complete

my s t u d i e s .

Discussion ............................... Sect ion I11 . I n h i b i t i o n of moulting i n Schis tocerca

g r e q a r i a i n f e c t e d by Mermis n iqrescens .

In t roduc t ion ............................. .................... Mater ia l s and Methods

Resul t s .................................. Discussion ...............................

........... Research Topics Ar i s ing ou t of t h i s P r o j e c t

............................................ References

.............................................. Appendix

...................................... Curriculum v i t a e

Page

52

v i i i

LIST OF TABLES

Page

Table I The e f f e c t of duration of storage a t 5 ' ~

on the v i a b i l i t y of f i f t y Mermis niqrescens

eggs i n 7 -day-old adul t Schistocerca

greqaria hosts . Locusts examined 25 days

a f t e r in fec t ion ......................... 1 9

Table I1 The e f f e c t of the infec t ive dose of

Mermis niqrescens on the duration of

the p a r a s i t i c phase. One -day-old

Schistocerca qreqaria adul t s infected,

...................... and reared a t 3 5 ' ~ 23

Table I11 The e f f e c t of rear ing temperature on the

duration of development of Mermis

niqrescens larvae i n 1-day-old

Schistocerca qreqaria adul t s infected

......................... with f i f t y eggs 2 4

Table I V The e f f e c t of the sex of Schistocerca

greqaria adul t s on the duration of the

p a r a s i t i c phase of Mermis niqrescens

................... larvae reared a t 3 5 ' ~ 27

Page

Table V Effect of sex of Schistocerca

qreqaria on the s i ze of Mermis

niqrescens larvae. Rearing tempera-

tu re , 35OC; infec t ive dose, f i f t y

eggs. ................................... 2 8

Table V I The e f f e c t of the age of Schistocerca

qreqaria on the duration of the para-

s i t i c phase of Mermis niqrescens larvae,

reared a t 35OC , with an infec t ive dose

of f i f t y eggs. ........................... 30

Table V I I The e f f e c t of t ransfer r ing Mermis

niqrescens larvae t o a second healthy

host (Schistocerca qreqaria)on the t o t a l

development time, mean length and mean

maximum width of the paras i te ........... 35

Table V I I I Effect of Mermis niqrescens parasi t ism on

the moulting of Schistocerca qreqaria

a t l e a s t s ixteen r ep l i ca t e s for each

Table IX

host age and infec t ive dose. ............ 64

Effect of Mermis niqrescens parcsi t ism on

the incorporation of leucine el4 i n t o

Page

t h e f a t body p r o t e i n s of Schis tocerca

q r e q a r i a . S ix r e p l i c a t e s per h o s t , and

........ s i x h o s t s f o r each incubat ion t ime 69

able X Amount of p r o t e i n r e l e a s e d i n t o t h e

incubat ion medium by f a t body from

c o n t r o l and i n f e c t e d f i f t h i n s t a r

Schis tocerca q r e q a r i a . S ix r e p l i c a t e s

pe r h o s t , and s i x h o s t s f o r each

.......................... incubat ion t ime 70

Table X I E f fec t of ~ e r m i s n iqrescens p a r a s i t i s m

14 on t h e incorpora t ion of l euc ine C i n t o

p r o t e i n s r e l e a s e d from t h e f a t body of

Schis tocerca q r e q a r i a . S ix r e p l i c a t e s per

h o s t , and s i x h o s t s f o r each incubat ion

................................... t ime. 71

LIST O F FIGURES

Page

Figure 1.

Figure 2 .

Figure 3 .

Diagrammatic representation of the

digestive system of Schistocerca

greqaria, showing l iga t ion s i t e s

(numbered one t o seven). Region

marked by shading indicates where

Mermis niqrescens eggs hatch and larvae

penetrate through the gut wall i n to

........................... the haemocoel 20

Effect of rearing temperature on the

irieaii ieiigiil and mean maximum w i d t h of

Mermi.s niqrescens larvae i n Schistocerca

qreqaria infected with f i f t y para-

s i t e eggs one day a f t e r the imaginal

moult ................................... 2 5

Effect of the age of Schistocerca

qreqaria on the mean length of Mermis

niqrescens larvae. Rearing temperature,

3 5 ' ~ ; infect ive dose, f i f t y eggs.. ...... 31

x i i

Page

Figure 4. E f f e c t of t h e age of Schis tocerca

q r e q a r i a on t h e mean maximum width

of Mermis n iqrescens l a r v a e . Rearing

temperature, 3 5' C ; i n f e c t i v e dose,

............................ f i f t y eggs. . 32

Figure 5 . E f f e c t of i n f e c t i v e dose on the sex

r a t i o of Mermis n iqrescens i n

................... Schis tocerca q r e q a r i a 50

Figure 6. E f f e c t of h o s t weight and i n f e c t i v e dose

on t h e sex r a t i o of Mermis niqrescens i n

Schis tocerca q r e q a r i a . Percent male

nematodes developing g iven f o r each l i n e . 51

Figure 7 . E f f e c t of i n f e c t i v e dose on Mermis

n iqrescens l a r v a l m o r t a l i t y i n

Schis tocerca q r e q a r i a ................... 53

Figure 8 . Musca domestica l a r v a e used i n t h e b io -

a s say of ecdysone l e v e l s dur ing t h e

imaginal moult i n h e a l t h y and i n f e c t e d

Schis tocerca q r e q a r i a ................... 60

x i i i

Page

Figure 9 . Mermithid-infected adul t Schistocerca

qreqaria, showing deformed wings

............... resu l t ing from parasitism 65

Figure 10. Effect of Mermis nigrescens parasitism

on the t o t a l haemolymph protein levels

i n f i f t h i n s t a r Schistocerca qreqaria,

infected with f i f t y paras i te eggs 1 day

a f t e r they had moulted i n t o the fourth

............... i n s t a r and reared a t 35OC 67

Figure 11. Effect of Mermis niqrescens parasit ism

on the t o t a l f a t body protein levels i n

f i f t h i n s t a r Schistocerca qreqaria,

infected with f i f t y paras i te eggs 1 day

a f t e r they had moulted i n t o the fourth

i n s t a r and reared a t 35•‹C... . . . . . . . . . . . . 67

x iv

GENERAL INTRODUCTION 0

Schis tocerca qreqaria Forskal . , the d e s e r t l o c u s t , has

been an important c rop p e s t f o r a t l e a s t 3000 yea r s throughout

t h e h o t t e r p a r t of t h e Old World inc lud ing most of Afr ica .

populat ions of t h e d e s e r t l o c u s t f l u c t u a t e i n numbers a s a

r e s u l t of t h e e f f e c t s o f many f a c t o r s inc luding those of

p reda to r s , p a r a s i t e s and microorganisms. However, t h e s e

f a c t o r s have been i n s u f f i c i e n t t o reduce t h e damage p o t e n t i a l

caused by 5, g r e q a r i a t o an ecocomically accep tab le l e v e l over

a long pe r iod . Those few r e p o r t s t h a t have claimed high mort-

a l i t y t o d e s e r t l o c u s t numbers have been t h e r e s u l t of labor-

a t o r y s t u d i e s and/or small f i e l d t e s t s . Although s e v e r a l

organisms show p o t e n t i a l i n c o n t r o l l i n g hlgh d e n s i t y l o c u s t

populat ions i n t h e f i e l d , q u a n t i t a t i v e e s t ima tes o f t h e i r

e f f e c t i v e n e s s i s l ack ing .

~ n t o m o p h i l i c nematodes have g r e a t p o t e n t i a l f o r use

a s b i o l o g i c a l c o n t r o l agents a g a i n s t a g r i c u l t u r a l i n s e c t p e s t s

(Welch, 1962a, 1965; Poinar , 1971; Nickle, 1972) . One family,

t h e Mermithidae, p a r a s i t i z e c rus taceans , s p i d e r s and s n a i l s

( C h a t t e r j e e and Singh, 1965) b u t , a r e p r imar i ly i n s e c t para-

s i t e s . Mermithids always k i l l t h e i r h o s t s upon emergence from

them. They occur a s i n t e r n a l l a r v a l p a r a s i t e s , but a s a d u l t s

a r e f r e e - l i v i n g i n s o i l o r f r e s h water . I n some spec ies ,

t h e l a r v a e grow up t o 15 cm i n l eng th . They d e r i v e t h e i r

- 2 -

nutrients from the host s haemocoel and subsequently cause

major changes in host metabolism, physiology and behavior

(Welch, 1965; Poinar, 1971) and thus, retard host development

and egg production (Strickland, 1911; Christie, 1936; Welch,

1960; Denner, 1968). Strickland (1911) noted abnormal organ

development and partial degeneration of the thoracic flight

muscles in mermithid infected blackflies (Diptera: Simuliidae) .

Welch (1960) reported total fat body depletion in mosquitoes

infected with Hydromermis churchilliensis. Earlier work •’re-

quently described suppression or resorption of infected host

oocytes (Christie, 1936, 1937 ; Crowcroft, 1948) . Phelps and

DeFoliart (1964) found total fat body depletion and oocyte

resorption in female hosts and suppression of testes in the

males in mermithid-parasitized blackflies. Poinar and Gyrisco

(1962) described similar changes in the alfalfa weevil attacked

by Hexamermis arvalis. A single mermithid worm invariably

kills the host when emerging from it, probably as a result of

the loss of host body fluids. Apart from the observed effects

of mermithids on host tissues, the most commonly manifested

result of mermithid parasitism is physiological castration of

the female hosts, Thus, mermithids have the potential of

regulating pest species by both rendering the host sterile and

eventually killing the survivors.

- 3 -

Since mermithids have a world-wide d i s t r i b u t i o n , t h e i r

presence i s coinc ident wi th t h a t of many s u s c e p t i b l e h o s t

i n s e c t s . Mermithids a r e o b l i g a t e p a r a s i t e s , a r e r e l a t i v e l y

h o s t s p e c i f i c and up t o 100% p a r a s i t e inc idence has been r e - . .

por ted (Sugiyama, 1956; Wulker, 1961; Welch, 1962b; Rubtsov,

1964; Welch and Rubtsov, 1965) . Although l i t t l e i s known

about t h e e f f e c t s of t h e phys ica l environment on s u r v i v a l of

mermithid eggs, some l a b o r a t o r y s t u d i e s i n d i c a t e t h a t prolonged

s t o r a g e i s f e a s i b l e (Pe te r sen , Chapman and Woodard, 1968;

Pe tersen , 1972) .

Although t h e r e a r e no records of n a t u r a l l y occurr ing

nematode p a r a s i t e s of t h e d e s e r t l o c u s t , t h e r e a r e s e v e r a l

r e p o r t s of t h e nat i ixal occurrence of roermithids among g rass -

hoppers ( C h r i s t i e , 1936, 1937; Bayl i s , 1944, 1947; Smith, 1944,

1947; Denner, 1968) . Mermis subniqrescens i s a common p a r a s i t e

o f grasshoppers i n t h e uni ted S t a t e s ( c h r i s t i e , 1937) . Gravid

females migra te t o t h e s o i l s u r f a c e , climb t h e vege ta t ion and

d e p o s i t t h e i r eggs, which are inges ted by feeding grasshoppers .

The eggs ha tch i n t h e a l imentary t r a c t o f t h e h o s t , and t h e

young l a r v a e p e n e t r a t e t h e g u t w a l l and develop i n t h e body

c a v i t y . The p a r a s i t e remains i n t h e h o s t from 4 t o 10 weeks.

P a r a s i t e s k i l l t h e h o s t on emergence and complete t h e remainder

of t h e i r development i n t h e s o i l . Denner (1968) observed a

- 4 -

d e c r e a s e i n egg produc t ion i n mermithid- infected grasshoppers

which he a t t r i b u t e d t o t h e presence of nematode exuda tes and

s e c r e t i o n s . The l e n g t h o f t h e M. subniqrescens p a r a s i t e was

dependent on h o s t s i z e and on t h e number o f p a r a s i t e s p e r h o s t .

p a t h o l o g i c a l e f f e c t s o f M. - subn iq re scens i n f e c t i o n a r e i n h i b i -

t i o n o f gonad development, s t e r i l i t y and longer nymphal i n s t a r s .

M. - subniqrescens i s an impor tan t f a c t o r i n g rasshopper c o n t r o l

th roughout t h e p a r a s i t e ' s h o s t r ange ( c h r i s t i e , 1937) .

The l i f e c y c l e o f g. n i q r e s c e n s i s v e r y s i m i l a r t o t h a t

o f g. subniqrescens . Both p a r a s i t e s have been r e p o r t e d a s

p l a y i n g an impor tan t n a t u r a l r o l e i n suppres s ing grasshopper

p o p u l a t i o n s ( C h r i s t i e , 1937; B a y l i s , 1944, 1947; Denner, 1968) ,

and &. n i q r e s c e n s h a s a l s o been r e p o r t e d t o p a r a s i t i z e t h e

earwig, ~ o r f i c u l a a u r i c u l a r i a (Crowcrof t , 1948) . M. n i s r e s c e n s

and g. subn iq re scens may be t h e s a m e s p e c i e s ( B a y l i s , 1947;

N ick le , 1972) o r may b e s e p a r a t e s p e c i e s (Cobb, 1926; Denner,

1968) . Gordon and Webster (1971) s t u d i e d t h e p h y s i o l o g i c a l

r e l a t i o n s h i p between g. n i q r e s c e n s and S . q r e q a r i a , an un-

n a t u r a l h o s t . M. n i q r e s c e n s p a r a s i t i s m caused a d e p l e t i o n

o f t o t a l haemolymph carbohydra tes i n male and female h o s t s

d u r i n g t h e t i m e when t h e most a c t i v e nematode growth was

o c c u r r i n g . Suppress ion of oocy te development and oocy te

r e s o r p t i o n were noted i n female h o s t s . Fa t body p r o t e i n s and

amino a c i d s were decreased i n i n f e c t e d h o s t s during t h e l a t t e r !

$ s t a g e s of i n f e c t i o n , and p a r a s i t i s m caused a s i g n i f i c a n t

r educ t ion i n t h e l e v e l of glycogen and non-glycogen carbohydrates

i n t h e h o s t f a t body (Gordon and Webster, 1971; Gordon, Webster

and Mead, 1972) . They hypothesized t h a t t h e nematode, by

feeding on t h e blood carbohydrates of the h o s t , caused decreased

glycogenesis i n t h e h o s t f a t body, thus depr iv ing t h a t organ

of carbohydrates .

Despite t h i s r e c e n t evidence, t h e r e remains s e v e r a l

important a s p e c t s of t h e h o s t - p a r a s i t e a s s o c i a t i o n t o be inves-

t i g a t e d before M. niqrescens can be c u l t i v a t e d i n v ivo f o r

use i n t h e b i o l o g i c a l c o n t r o l of 2. g r e g a r i a . Therefore, my

o b j e c t i v e s were: I . To s tudy t h e development of M. niqrescens

i n 5. qreqar i a i n t h e l abora to ry . 11. To determine t h e i n t e r -

a c t i o n between M. niqrescens and 2. q r e q a r i a , and 111. To

determine t h e mechanisms of M. niqrescens pa thogenic i ty on

S. q r e q a r i a . -

- 6 -

GENERAL MATERIALS AND METHODS

1, Rearinq of Schis tocerca qr-eqaria -

The l o c u s t colony, obtained i n i t i a l l y from t h e Univers i ty

of B r i t i s h Columbia, was r e a r e d i n t h e i n s e c t a r y under con-

s t a n t l i g h t a t a temperature of 34-36 '~ , a r e l a t i v e humidity

of 60-70%, and on a mixed d i e t of whole wheat bran and l ab -

oratory-grown g r a s s . The d i e t w a s chosen s o as t o preclude

t h e p o s s i b i l i t y of exposing t h e l o c u s t s t o a n a t u r a l mermithid

i n f e c t i o n . The d i e t throughout t h e s tudy remained t h e same.

The bran mixture was composed of 6 p a r t s whole wheat bran,

2 p a r t s g r a s s meal, 1 p a r t white brewer 's y e a s t and 1 p a r t

. A 1 4 - 1 O C C \ y u r r u b r ~ u A u r r r k u L u u , I J J U ) .

I n a l l experiments, male and female l o c u s t s were main-

t a i n e d i n s e p a r a t e cages.

2 . Co l l ec t ion of Mermis n iqrescens eqqs

Adult M. niqrescens females were c o l l e c t e d from a newly

1 ploughed f i e l d i n Chill iwack , B r i t i s h Columbia, i n e a r l y

June of 1970, 1971, and 1972. The nematodes were found on t h e

1 Col lec t ions made by cour tesy of M r . E . Bollerup, 50105 Pa t t e r son Road, Chill iwack, B r i t i s h Columbia.

s o i l su r face e i t h e r dur ing o r immediately a f t e r a period of

e a r l y morning r a i n . The females which emerged from t h e s o i l

t o commence egg l ay ing on t h e nearby f o l i a g e were c o l l e c t e d

and placed on a moistened, f i l t e r - p a p e r - l i n e d , P e t r i d i s h i n

which they l a i d eggs. The i n f e c t i v e eggs were washed t h r e e

t imes wi th s t e r i l e water and cen t r i fuged a t low speeds a f t e r

each washing t o remove d e b r i s and su r face contaminants, and

then s t o r e d d r y on f i l t e r - p a p e r - l i n e d P e t r i d i shes a t 5 ' ~ u n t i l

r equ i red .

3 . I n f e c t i o n of Schis tocerca q r e q a r i a wi th mermithid eqqs

Mermithid eggs were t r a n s f e r r e d , using a f ine- t ipped

disposable Pasteur pipette under a sterensropic mi~roscnpe:

onto small p ieces of laboratory-grown g r a s s coated wi th a t h i n

l a y e r of a Gelgard M - water suspension (Webster and Bronsk i l l ,

1968) t o f a c i l i t a t e adhesion of t h e eggs. This enabled a pre-

c i s e dosage of M_. niqrescens eggs t o be g iven t o each l o c u s t .

Locusts, pres tarved f o r 4 hours , were fed t h e mermithid-

i n f e c t e d g r a s s . Non-infected c o n t r o l groups were s e t up i n

sepa ra te cages f o r each experiment.

- 8 -

4 , s t a t i s t i c a l a n a l y s i s o f r e s u l t s

The d a t a p re sen ted i n t h i s study w e r e analyzed s t a t -

i s t i c a l l y u s ing S t u d e n t ' s t - t e s t , w i t h s i g n i f i c a n c e a t t h e

0.05 (5%), 0.01 (1%) and 0.001 (0.1%) l e v e l s be ing accep ted

and i n d i c a t e d i n t h e t a b l e s and f i g u r e s by t h e symbols

a , b and c a s fo l lows:

S i g n i f i c a n c e a t t h e 0.05 (5%) l e v e l .... a

S i g n i f i c a n c e a t t h e 0.01 (1%) l e v e l .... b

S i g n i f i c a n c e a t t h e 0.001 (0.1%) l e v e l . . c

SECTION I

The e f f e c t s of s e l e c t e d e x t e r n a l f a c t o r s and t h e age, sex ,

and n u t r i t i o n a l s t a t e of the d e s e r t l o c u s t on t h e development

of Mermis n iqrescens .

- 10 - i NTRODUCTION

The eggs of M. niqrescens , unl ike those of most o t h e r

mermithids (Phelps and DeFol iar t , 1964; Trp i s , Haufe and

Shemanchuk, 19681, a r e swallowed by the hos t dur ing feeding

and they then ha tch i n t h e g u t and t h e l a rvae p e n e t r a t e i n t o

t h e haemocoel. Previously documented s t u d i e s on t h e develop-

ment of M. niqrescens , however, a r e unclear a s t o t h e develop-

ment of t h e p a r a s i t e dur ing t h e f i r s t 4 days a f t e r i n f e c t i o n .

Of p a r t i c u l a r importance i s t h e f a c t t h a t t h e r e i s inconclusive

evidence a s t o t h e s i t e of egg ha tching and subsequent g u t

w a l l pene t ra t ion . C h r i s t i e (1937) s t a t e s t h a t t h e eggs hatched

1-1 1/2 hours a f t e r i n g e s t i o n by t h e h o s t and t h a t t h e l a rvae

penet ra ted t h e i n t e s t i n a l wa l l of t h e grasshopper , Melanoplus

femur-rubrum ( ~ e G e e r ) 20 minutes l a t e r . Bayl is (1947 )

found t h a t when t h e eggs of M, niqrescens i n f e c t F. a u r i c u l a r i a ,

they ha tch i n t h e g u t , and t h e l a r v a e a c t i v e l y migrate through

the gut wa l l i n t o t h e body c a v i t y . I n s i d e the haemocoel, t he

l a r v a e changed l i t t l e f o r 2 days, except t h a t t h e c e l l u l a r

con ten t s of the body showed s i g n s of being arranged i n a long i -

t u d i n a l s e r i e s of i r r e g u l a r , s o l i d masses. Nei ther the r e g i o n ( s )

of t h e g u t where pene t ra t ion of the M. niqrescens l a rvae i n t o

t h e haemocoel occurs nor t h e f a c t o r ( s ) t h a t govern egg hatching

have been e l u c i d a t e d .

- 11 -

There i s l i t t l e information on t h e v i a b i l i t y of

mermithid eggs under l abora to ry cond i t ions . Pe tersen e t a 1 . ,

(1968) found t h a t t h e eggs of Romanomermis s p . , which para-

s i t i z e t h e a q u a t i c l a rvae of S imul i ids , could be s t o r e d i n sand

moistened wi th d i s t i l l e d water f o r a t l e a s t 6 months a t

ambient temperatures w i t h no s i g n i f i c a n t l o s s i n egg v i a b i l i t y .

Muspratt (1965) noted t h a t t h e eggs of an u n i d e n t i f i e d mermithid

p a r a s i t e of t h e mosquito, Culex p i p i e n s , survived s to rage i n

t h e l a b o r a t o r y f o r s e v e r a l months i f t h e eggs were placed i n

a box of sand of h igh water con ten t and s lowly des icca ted .

There i s no publ ished record of long-term s to rage of M. -

niqrescens eggs i n t h e l abora to ry .

The s i z e of t h e g. niqrescens l a rvae upon emergence from

t h e h o s t has been pos tu la ted a s being governed by t h e i n i t i a l

i n f e c t i v e burden ( C h r i s t i e , 1929; Bayl is , 1944; Denner, 1968) . Gupta and Burr (unpublished observat ion) have shown t h a t t h e

i n f e c t i v e dose of eggs and the h o s t ca tegory (by age and

weight) of - S. q r e q a r i a govern t h e emergence s i z e of t h e M. -

niqrescens l a r v a e . C h r i s t i e (1937) claims t h a t M. subniqrescens

l a rvae grew l a r g e r and remained longer i n a l a r g e grasshopper

than i n a small one, and a l s o t h a t t h e fewer nematodes t h a t a

grasshopper harboured, the l a r g e r these worms became and t h e

longer they remained wi th in the h o s t . Other r e s e a r c h e r s , using

a v a r i e t y of o t h e r h o s t s of mermithids, have reached s i m i l a r

~ o n c l u s i o n s ( S t a b l e r , 1952; Welch, 1960, 1965; Denner, 1968;

Trpis e t a l , , 1968; Pe tersen and W i l l i s , 1970) .

Severa l r e p o r t s have i n d i c a t e d t h a t mermithid p a r a s i t e s

a r e more o f t e n found i n one developmental s t a g e of t h e hos t

than i n another ( C h r i s t i e , 1936, 1937; Welch, 1960; Poinar and

~ y r i s c o , 1962; Pe tersen et a,, 1968; Pe tersen and W i l l i s , 1970) .

Whether one developmental s t a g e of S. q r e q a r i a i s more s u s c e p t i b l e

t o M. niqrescens p a r a s i t i s m than another s t a g e i s no t known.

There a r e c o n f l i c t i n g r e p o r t s i n t h e l i t e r a t u r e on

t h e e f f e c t of t h e sex of t h e h o s t on t h e development of t h e

mermithid p a r a s i t e . Pa ras i t i sm of t h e grasshoppers Melanoplus

a t l a n i s and M_. b i v i t t a t u s by t h e nematode Mermis f e r r u q i n i a

r e s u l t e d i n 45% of t h e female h o s t s being i n f e c t e d b u t only

9% of t h e males (Glaser and Wilcox, 1918) . C h r i s t i e ( l 9 3 6 ) ,

however, found no s i g n i f i c a n t d i f f e r e n c e i n percent p a r a s i t i s m

of male and female h o s t s i n s e v e r a l s p e c i e s of short-horned

grasshoppers (Orthoptera: Acr id idae) when i n f e c t e d wi th A_.

decaudata .

I n order t o c l a r i f y some of t h e confusion i n the l i t e r a -

t u r e , a s e r i e s o f experiments were undertaken using g. niqrescens

i n t h e d e s e r t l o c u s t under c o n t r o l l e d cond i t ions . An exper i -

ment was s e t up t o determine the s i t e of p a r a s i t e egg ha tching

and subsequent l a r v a l p e n e t r a t i o n i n t o the h o s t haemocoel and

t h e f a c t o r s t h a t c o n t r o l t h e s e even t s . The e f f e c t s of long-

term s to rage of - M. n iqrescens eggs on t h e i r v i a b i l i t y and t h e

temperature a t which p a r a s i t e development occurs most r a p i d l y

a r e two a r e a s about which nothing i s known, y e t which must be

e luc ida ted before M. - niqrescens can be mass produced i n t h e

l abora to ry . E f f e c t s of p a r a s i t e burden, age, sex and weight

of t h e d e s e r t l o c u s t on t h e d u r a t i o n of - M. niqrescens develop-

ment and on t h e s i z e of t h e nematode l a rvae upon emergence from

t h e h o s t a r e a l s o a r e a s which must be c l a r i f i e d . The r e s u l t s

of t h e s e i n v e s t i g a t i o n s a r e presented i n t h i s s e c t i o n .

MATERIALS AND METHODS

1. - M. n iqrescens eqq v i a b i l i t y

Th i r ty - f ive , 7 -day-old a d u l t female l o c u s t s were each

i n f e c t e d wi th f i f t y nematode eggs, d i s s e c t e d 25 days l a t e r and

t h e t o t a l number of developing nematode l a r v a e i n the

haemocoel counted. g. niqrescens eggs which had been s to red

f o r 3 , 15 , 27 and 39 months a t 5 ' ~ and 55-60% R. H. were used

t o i n f e c t the l o c u s t s .

2 . Development of the p a r a s i t i c phase of M. n iqrescens -

A l l developmental s t u d i e s were done using 1-day-old

a d u l t male l o c u s t s a s h o s t s , each i n f e c t e d wi th f i f t y eggs.

~ l l measurements were done using a d i s s e c t i n g microscope and

an ocu la r micrometer. I n growth de terminat ions , each l o c u s t

was d i s s e c t e d i n i n s e c t s a l i n e s o l u t i o n (Appendix I ) a t d i f -

f e r e n t t imes a f t e r i n f e c t i o n . The l a r v a e from each h o s t were

sexed and then h e a t - k i l l e d p r i o r t o measuring. Nematodes t h a t

had developed f o r l e s s than 7 days were processed a s descr ibed

below.

To determine egg ha tching t ime, 3. niqrescens eggs

were dipped i n a 1% s o l u t i o n of Sudan black o r methylene blue

2 - - T n ~ l 7 c i - c and the excess s t a i n removed by three water washinnc ,,,,,,,

were k i l l e d a t d i f f e r e n t t imes a f t e r i n f e c t i o n , ranging from

20 minutes t o 2 4 hours . A t each time i n t e r v a l , t h e g u t was

removed and t h e t o t a l number of egg cas ings recorded. To

i d e n t i f y t h e g u t r eg ion i n which most of t h e eggs hatched,

l o c u s t s i n f e c t e d wi th f i f t y eggs were d i s s e c t e d immediately

from t h e v e n t r a l su r face i n i n s e c t s a l i n e s o l u t i o n and the gu t

l i g a t u r e d i n one of the fol lowing p laces : - Ante r io r end of t h e

esophagus, p o s t e r i o r end of t h e esophagus, p o s t e r i o r end of

the crop , p o s t e r i o r end of t h e p roven t r i cu lus , p o s t e r i o r t o t h e

po in t of o r i g i n of t h e Malpighian tubu les , midway along t h e

hindgut and a t the junct ion of t h e hindgut and rectum (See Figure I ) .

Each s e c t i o n of t h e g u t was d i s s e c t e d open e i t h e r 7 , 1 2 , o r

2 4 hours a f t e r l i g a t u r i n g , and t h e number of egg cas ings

counted. Seventy l o c u s t s were used i n t h e s tudy.

Nematode development up t o Day 4 of i n f e c t i o n was

s t u d i e d by two methods. Two hours a f t e r i n f e c t i o n , l o c u s t s

were d i s s e c t e d and the g u t l i g a t u r e d a n t e r i o r t o t h e pharynx

and p o s t e r i o r t o t h e rectum. The e n t i r e g u t was then placed

i n i n s e c t s a l i n e s o l u t i o n i n a g l a s s P e t r i d i s h . Passage of

nematodes through t h e g u t w a l l was observed under t h e com-

pound microscope using t r ansmi t t ed l i g h t . I n t h i s experiment,

t h i r t y - s i x l o c u s t s were used. I n another experiment, i n f e c t e d

l o c u s t s were decap i t a t ed a t i n t e r v a l s from 6 hours t o 5 days

a f t e r i n f e c t i o n , t h e a n t e r i o r of t h e g u t l i g a t u r e d , and t h e

e n t i r e l o c u s t body cent r i fuged a t low speed t o e x t r a c t t h e

i n s e c t haemolymph and nematode l a r v a e . Measurements of the

mean l eng th and mean maximum width of t h e l a rvae were made a s

previous ly . I n some i n s t a n c e s , t h e a d d i t i o n of 0.5% Cotton

b lue f a c i l i t a t e d l o c a t i o n of l a rvae i n t h e e x t r a c t e d haemo-

lymph. Twenty-nine l o c u s t s were used i n t h i s experiment.

The e f f e c t of p a r a s i t e burden on nematode develop-

ment was s t u d i e d i n 1-day-old a d u l t m a l e and female l o c u s t s

i n f e c t e d wi th t e n , t h i r t y o r f i f t y nematode eggs. The mean

number of emergent nematode l a r v a e and t h e dura t ion of t h e i r

p a r a s i t i c development were observed f o r each l o c u s t . Between

t h r e e and s i x l o c u s t s were used i n each s tudy.

A l l h o s t s used i n temperature e f f e c t s t u d i e s were

1-day-old a d u l t s of both sexes . Each l o c u s t was i n f e c t e d wi th

f i f t y nematodes and rea red a t 2 2 ' ~ o r 3 5 ' ~ . The mean number

of emergent l a r v a e , t h e i r mean p a r a s i t i c development t ime and

t h e i r mea.1 l eng th and mean maximum width were recorded.

The e f f e c t s of h o s t sex on M. - niqrescens development

was s tud ied using twenty, 1-day-old a d u l t l o c u s t s o f each sex

and twenty, 28-day-old a d u l t l o c u s t s of each sex. Each h o s t

was i n f e c t e d wi th t e n , t h i r t y o r f i f t y nematode eggs each,

r e s p e c t i v e l y , and reared a t 2 2 ' ~ o r 3 5 ' ~ .

The e f f e c t of h o s t age on M. - niqrescens was s tud ied

i n l o c u s t s of f i v e ages: One-day-old t h i r d i n s t a r nymphs,

4-day-old four th i n s t a r nymphs, 5-day-old f i f t h i n s t a r nymphs,

1-day-old a d u l t male and female l o c u s t s and 28-day-old a d u l t

male and female l o c u s t s . Twenty l o c u s t s from each group and

sex were reared i n s e p a r a t e cages. Each h o s t was fed f i f t y

p a r a s i t e eggs and t h e mean number o f emergent l a r v a e , t h e i r

development t ime and t h e i r mean l eng th and mean maximum width

were recorded.

E f f e c t of t r a n s f e r r i n q M . n igrescens l a r v a e t o a second -

hos t on t h e t o t a l development time and t h e s i z e of t h e

p a r a s i t e .

Twenty-five days a f t e r i n f e c t i o n of twelve, 1-day-old

a d u l t male and female l o c u s t s wi th f i f t y nematode eggs, the h o s t s

were k i l l e d , the nematodes removed, and t h e i r mean l eng ths and

mean maximum widths recorded. Uninfected 1-day-old a d u l t

l o c u s t s were anaes the t i zed i n CO and one, t h r e e , f i v e o r t e n 2 ' -

nematodes i n s e r t e d i n t o t h e haemocoel through a 5 mm abdominal

i n c i s i o n . The i n c i s i o n was daubed wi th chloramphenicol c r y s t a l s

to s t o p i n f z z t i ~ n azd t he2 sezled w i t h zelted parazf in wax.

One s e t of c o n t r o l s cons i s t ed of 1-day-old a d u l t l o c u s t s i n -

f ec ted wi th e i t h e r one, t h r e e , f i v e o r t e n nematodes. The t o t a l

development time and t h e mean l eng th and mean maximum width of

emergent worms were recorded. A second s e t of c o n t r o l s were

sham operated b u t not i n f e c t e d .

RESULTS

1. M. n iqrescens eqq v i a b i l i t y -

Eggs s t o r e d d ry a t ~ O C and 55-60% R.H. f o r up t o

15 months showed no s i g n i f i c a n t l o s s i n v i a b i l i t y , a s i n -

d i c a t e d by t h e number of nematode l a r v a e found i n t h e haemocoel

(Table I ) . Eggs s t o r e d f o r 27 o r 3 9 months under s i m i l a r

(p re - in fec t ion) cond i t ions showed a s i g n i f i c a n t (P<O .001)

decrease i n v i a b i l i t y . The longer t h e time of s t o r a g e , t h e

g r e a t e r was the range of v i a b i l i t y of t h e eggs from h o s t t o

h o s t . Eggs used a f t e r 39 months s to rage ranged i n v i a b i l i t y

from 20-60%) a s opposed t o 85-95% a f t e r 3 months.

2 . Development of the p a r a s i t i c phase of M. n igrescens -

The nematode eggs hatched i n t h e r eg ion of t h e

h o s t g u t between t h e p o s t e r i o r end of t h e crop and a n t e r i o r

t o the p o i n t of o r i g i n of t h e Malpighian tubu les 3 1/2 t o 6 hours

a f t e r

t h a t

i n g e s t i o n (Fig. 1).

the ha tching of the

Liga tur ing experiments ind ica ted

n iqrescens i n the d e s e r t

l o c u s t g u t was s i t e s p e c i f i c . Eggs d id no t ha tch i n o the r

p a r t s of t h e g u t , even 24 hours a f t e r i n f e c t i o n . When a

l i g a t u r e was made a t the p o s t e r i o r end of t h e crop immediately

a f t e r feeding t h e eggs t o t h e h o s t , i n t a c t eggs

TABLE I

The e f f e c t of dura t ion of s to rage a t 5 ' ~ on t h e v i a b i l i t y of f i f t y Mermis niqrescens eggs i n 7-day-old a d u l t Schis tocerca q r e q a r i a h o s t s . Locusts examined 2 5 days a f t e r i n f e c t i o n .

- period of No. Locusts Mean Number of P a r a s i t e s

Examined Developing i n t h e Host +_ S , E . 1

s to rage

3 months 10

15 months

27 months

39 months

1 Means followed by t h e same l e t t e r a r e s i g n i f i c a n t l y d i f f e r e n t .

Figure 1. Diagrammatic r e p r e s e n t a t i o n of t h e d i g e s t i v e system

of Schis tocerca q r e s a r i a , showing l i g a t i o n s i t e s

(numbered one t o seven) . Region marked by shading

i n d i c a t e s where Mermis n iqrescens eggs ha tch and

l a r v a e p e n e t r a t e through t h e g u t wa l l i n t o t h e

haemocoel.

ESOP

HAGU

S

MO

UTH

GIZZ

ARD

I (P

ROVE

NTRI

CULU

S)

MID

GUT

but no empty egg casings were recovered an ter ior t o the

l iga tu re e i t h e r 7 or 24 hours a f t e r feeding. Similarly, when

a l iga ture was made midway along the hindgut immediately

a f t e r infect ion and the gut was dissected 12 hours l a t e r , the

majority of empty egg casings and a l l of the nematode larvae

detected were observed i n the midgut. When l iga tures were

made a t posit ions three and f ive (Fig. 1) and 1 2 hours l a t e r

the l iga tu re a t posit ion three was severed, a l l the empty egg

casings were found i n the midgut of the host upon dissect ion

4 hours l a t e r . Since the locust faeces were not examined for

egg casings, i t was not possible t o determine what percentage

of the 8. niqrescens eggs f a i l ed t o hatch.

Penetration of M_. niqrescens larvae from the gut

i n t o the haemocoel occurred i n the same region as egg

hatching, and from 1 t o 20 hours l a t e r . Pa r t i a l e x i t of the

nematodes from the i so la ted , infected guts i n t o insec t sa l ine

solut ion was observed on several occasions, but complete e x i t

from the gut was never observed. Newly hatched larvae i n the

gut possessed a s t y l e t , but it was not possible t o discern

,' whether gut wall penetration was i n t e r c e l l u l a r or i n t r a -

c e l l u l a r .

Centrifugation of decapitated locusts t o ex t rac t the

haemolymph plus nematode l a r v a e enabled almost complete r e -

covery of t h e young l a rvae from the haemocoel. Larvae ap-

peared i n the haemolymph 48 t o 72 hours a f t e r i n g e s t i o n of t h e

eggs by t h e d e s e r t l o c u s t . Very l i t t l e growth o r d i f f e r e n t -

i a t i o n of t h e l a r v a e occurred dur ing the first 4 days a f t e r

i n f e c t i o n .

A s t h e i n f e c t i v e dose of g. niqrescens eggs was i n -

c reased , t h e dura t ion of t h e p a r a s i t i c phase was s i g n i f i c a n t l y

shortened i n both male and female h o s t s (Table 11). There was

no s i g n i f i c a n t d i f f e r e n c e i n t h e percentage of nematodes t h a t

emerged from each h o s t i n f e c t e d wi th d i f f e r e n t numbers of

nematode eggs (Table 11) .

I n 1-day-old a d u l t male, b u t not female h o s t s , t h e

dura t ion of t h e p a r a s i t i c phase was s i g n i f i c a n t l y s h o r t e r a t

35' c than a t 22' c (Table 111) . There was no s i g n i f i c a n t

e f f e c t of r e a r i n g temperature on t h e t o t a l number of emergent

p a r a s i t e s (Table 111). Although t h e nematode's development

time was s h o r t e r a t 3 5 ' ~ than a t 22OC, t h e r e was no s i g n i f i c a n t

d i f f e r e n c e i n t h e i r d a i l y growth p a t t e r n s i n h o s t s of e i t h e r

sex (Fig. 2 ) ( a s based on mean l eng th and mean maximum width) .

The g r e a t e s t inc rease i n l eng th of t h e developing l a rvae

occurred between Days 11 and 1 7 a f t e r egg i n g e s t i o n when a

nine t o ten- fo ld i n c r e a s e occurred. S i m i l a r l y , t h e

TA

BL

E I1

The

e

ffe

ct

of

the

in

fec

tiv

e d

ose

o

f M

~r

rn

is

nic

rre

sce

ns

on

the

du

rati

on

of

the

pa

ras

itic

p

ha

se.

On

e-d

ay-o

ld

Sc

his

toc

erc

a g

req

ari

a a

du

lts

in

fec

ted

, an

d reared

at

35'~.

Sex

o

f N

o.

Lo

cu

sts

Infe

cti

ve

M

ean

No.

L

arv

ae

Mea

n D

ura

tio

n o

f P

ara

sit

ic

Ho

st

Infe

cte

d

Dose

E

mer

gin

g +

S .E.

Ph

ase

(

~a

ys

) +

s.

E.

~

Mal

e

Mal

e

Mal

e

Fem

ale

Fem

ale

Fem

ale

Mea

ns

foll

ow

ed

by

the

sa

me

lett

er

are

sig

nif

ica

ntl

y d

iffe

ren

t.

TA

BL

E I11

The

e

ff

ec

t o

f re

ari

ng

te

mp

era

ture

on

th

e d

ura

tio

n o

f d

ev

elo

pm

en

t o

f M

erm

is

niq

resc

en

s -

"la

rva

e i

n 1

-day

-old

S

ch

isto

ce

rca

qre

qa

ria

a

du

lts

in

fec

ted

wit

h f

if

ty

eg

gs.

Mea

n D

ura

tio

n o

f S

ex

of

No.

L

oc

ust

s R

ea

rin

g

Mea

n N

o.

Larv

ae

Dev

elo

pm

ent

Ho

st

Infe

cte

d

Te

mp

era

ture

E

mer

gin

g _

+ S

.E .

(~

ay

s)

_+

S

.E. 1

Mal

e

Mal

e

Fem

ale

Fem

ale

'~eean

s fo

llo

we

d b

y

the

sam

e le

tte

r a

re

sig

nif

ica

ntl

y d

iffe

ren

t.

Figure 2 . E f f e c t of r e a r i n g temperature on t h e mean l e n g t h

and mean maximum width of Mermis n iqrescens l a rvae

i n Schis tocerca q r e q a r i a i n f e c t e d wi th f i f t y para-

s i t e eggs one day a f t e r t h e imaginal moult.

v e r t i c a l b a r s r e p r e s e n t t 2 s tandard e r r o r s .

Mean l eng th of B. niqrescens l a r v a e i n a male

h o s t ; r e a r i n g a t 2 2 ' ~ and 3 5 ' ~ .

Mean maximum width of g. niqrescens l a r v a e i n a

male h o s t ; r e a r i n g a t 2 2 ' ~ and 3 5 ' ~ .

Mean l e n g t h of g. niqrescens l a r v a e i n a female

h o s t ; r e a r i n g a t 2 2 " ~ and 3 5 ' ~ .

Mean maximum width of M. niqrescens l a rvae i n a

female h o s t ; r e a r i n g a t 22Oc and 35Oc.

DAYS AFTER INGESTION OF EGGS


1

0 5 8 11



g r e a t e s t inc rease i n maximum width occurred between Days 11

and 14 a f t e r egg i n g e s t i o n , a 2 1/2-fold inc rease a t both

r e a r i n g temperatures .

Development time of g. niqrescens l a rvae was s i g n i f -

i c a n t l y longer i n female than i n male h o s t s i n f e c t e d e i t h e r

1 o r 28 days a f t e r t h e imaginal moult wi th f i f t y (but no t

t e n o r t h i r t y ) p a r a s i t e eggs and reared a t 3 5 ' ~ (Table I V ) . The mean body l eng ths of t h e nematodes i n female h o s t s of

e i t h e r i n f e c t i o n age were o f t e n s i g n i f i c a n t l y longer than

those i n male h o s t s of t h e same age (Table V ) . Host sex

had no s i g n i f i c a n t e f f e c t on p a r a s i t e width a t any time

dur ing l a r v a l development.

The age of t h e h o s t s i g n i f i c a n t l y a f f e c t e d t h e r a t e

of development of t h e g. niqrescens l a r v a e w i t h i n l o c u s t s

i n f e c t e d wi th f i f t y nematode eggs. The t o t a l development

time of t h e l a r v a e w i t h i n a t h i r d i n s t a r h o s t w a s s i g n i f i c a n t l y

s h o r t e r than w i t h i n e i t h e r a f o u r t h o r f i f t h i n s t a r h o s t under

i d e n t i c a l experimental cond i t ions (Table vI) . P a r a s i t e s

remained w i t h i n 28-day-old a d u l t male and female l o c u s t s

s i g n i f i c a n t l y longer than i n 1-day-old a d u l t s .

The comparative growth r a t e of g, niqrescens

changed wi th age of t h e h o s t , w i t h t h e r ap id growth t 7

per iods

d i f f e r e n c e s

TABLE

IV

The

e

ff

ec

t o

f th

e s

ex

of

Sc

his

toc

erc

a -

gre

c~

ari

a ad

ult

s o

n

the

du

rati

on

of

the

pa

ras

itic

p

ha

se

of

Mer

mis

n

igre

sc

en

s

larv

ae

re

are

d a

t 35'~.

No.

o

f M

ean

#

Em

erg

ing

M

ean

Tim

e o

f H

ost

s In

fec

tiv

e

fro

m

Em

erg

ence

Sex

Infe

cte

d

Dose

H

ost

+. S

.E.

(~

ay

s)

+_

S.E

. 1

Age

1-d

ay

-old

A

du

lt

1-d

ay

-old

A

du

lt

28

-day

-old

A

du

lt

28

-day

-old

A

du

lt

1-d

ay

-old

A

du

lt

1-d

ay

-old

A

du

lt

1-d

ay

-old

A

du

lt

1-d

ay

-old

A

du

lt

male

fe

ma

le

male

fe

ma

le

male

fe

ma

le

male

fe

ma

le

Mea

ns

foll

ow

ed

by

th

e s

ame

lett

er

ar

e s

ign

ific

an

tly

dif

fere

nt.

TA

BL

E

V

Effe

ct

of

sex

of

Sc

his

toc

erc

a q

req

ari

a on

the

s

ize

of

Me

rmis

niq

resc

en

s la

rva

e.

Re

ari

ng

te

mp

era

ture

,

35

'~;

infe

cti

ve

do

se

, f

if

ty

eg

gs.

Ho

st

1-D

ay-O

ld

Ad

ult

At

In

fec

tio

n

Ho

st

28

-Day

s-O

ld

Ad

ult

At

Infe

cti

on

Me

rmis

niq

resc

en

s

Me

rmis

niq

resc

en

s

Day

S

ex

Mea

n M

ean

- .M

e a

n

Mea

n A

fte

r

of

Le

ng

th 2

S.E

. 1

Max

. W

idth

+

S.E

. L

en

gth

+ S

.E.l

M

ax.

Wid

th 2 S

.E.

-

Infe

cti

on

H

os

t (m.. >

(mm. >

(m

e >

(m

.

Day

5

Da

y 5

Da

y 8

D

ay

8

Da

y 1

1

Da

y 1

1

Da

y 1

4

Da

y 1

4

Da

y 1

7

Da

y 1

7

Da

y 2

1

Da

y 2

1

ma l

e

fem

ale

ma

le

fem

ale

ma

le

fem

ale

ma

le

fem

ale

ma

le

fem

ale

ma

le

fem

ale

co

nti

nu

ed

.. .

.

Ta

ble

V

(c

on

tin

ue

d)

~o

st

1

-Day

-Old

A

du

lt A

t I

nfe

cti

on

H

ost

2

8-D

ays-

Old

A

t I

nfe

cti

on

Me

rmis

nig

res

ce

ns

M

erm

is n

iqre

sc

en

s

Day

Sex

Mea

n M

ean

Mea

n M

ean

Afte

r

of

Le

ng

th

+ S.E.

-

ax.

Wid

th

+ s

.E.

Le

ng

th

+ S .E

M

ax.

Wid

th +

S .E

. -

In

fe

cti

on

~

os

t

(m

. > (m

m.

1 (m

m-)

(m

. 1

Day

2

6

ma

le

---

---

---

---

10

2.0

0

25

.2

1

0.2

72

+

0.0

62

D

ay

26

fe

ma

le

10

1.4

0 +

7 -

21

0

.27

6 +

0.0

19

1

06

.39

+

6 .0

3

0.2

91

z 0

.03

0

Day

2

8

ma

le

---

---

---

---

10

7.1

0 +

4.0

0 b

0.2

90

2 0

.02

4

I

Day

3

6

fem

ale

--

- --

- --

- --

- 1

23

-65 +

9.2

2 b

0.3

02

2 0

.00

9

N a

I

Mea

ns

wit

hin

ea

ch

co

up

let

foll

ow

ed

by

t

he

sa

me

le

tte

r a

re

sig

nif

ica

ntl

y d

iff

er

en

t.

TAB

LE

VI

The

e

ff

ec

t o

f th

e a

ge

o

f S

ch

isto

ce

rca

qre

qa

ria

o

n

the

du

rati

on

of

the

pa

ra

sit

ic p

ha

se

of

Me

rmis

nis

res

ce

ns

la

rva

e,

rea

red

at 3

5'~

.,

wit

h a

n i

nfe

cti

ve

do

se o

f f

ifty

eg

gs.

Sc

his

toc

erc

a g

req

ari

a

Mer

mis

n

isre

sc

en

s

Age

Sex

No.

o

f H

ost

s M

ean

# E

mer

gin

g

Mea

n T

ime

of

at

In

fec

tio

n

Infe

cte

d

fro

m H

ost

+

S.E

.' E

merg

en

ce(D

ay

s)

+ S

.E. 1

-

-

1-d

ay

-old

A

du

lt

male

28

-day

-old

A

du

lt

male

1 -d

ay

-old

A

du

lt

fem

ale

28

-day

-old

A

du

lt

fem

ale

2 -d

ay

-old

5

th

---

Ins

tar N

ymph

4-d

ay

-old

4

th

Ins

tar N

ymph

1 -d

ay

-old

3

rd

Ins

tar

Nym

ph

- -- --

Mea

ns

foll

ow

ed

by

th

e

sam

e le

tte

r a

re s

ign

ific

an

tly

dif

fere

nt.

2 S

ign

ific

an

tly

dif

fe

re

nt

(p<

0.0

01

) fr

om

th

e n

um

ber

o

f g. n

iqre

sc

en

s

larv

ae

em

erg

ing

fr

om

ho

sts

of

al

l o

the

r a

ge

s.

Figure 3 . Effect of the age of Schistocerca qreqaria on the

mean length of Mermis niqrescens larvae. Rearing

temperature, 35OC; in fec t ive dose, f i f t y eggs.

Vert ical bars represent 2 2 standard e r ro r s .


16 2 0 24 28 32


Figure 4. Effec t of the age of Schistocerca qreqar ia on the

mean maximum width of Mermis n igrescens larvae.

Rearing temperature, 35'C; i n f ec t ive dose, • ’ i f t y

zggs .


I I I I I I I I

4 8 12 16 20 24 28 32 36


- 33 -

w i t h r e s p e c t t o t h e age of t h e h o s t were found a t any day sub-

sequent t o Day 5 a f t e r egg i n g e s t i o n . A t 14 days a f t e r egg

i n g e s t i o n , f o r example, nematodes ranged i n l eng th from 55.30

mm i n a d u l t males 1-day-old a t time of i n f e c t i o n t o 75.09 mrn

i n a d u l t females 28-days-old a t time of i n f e c t i o n . I n

nymphal h o s t s , nematode l eng ths ranged from 2.83 mm t o 21.90

mm; s i g n i f i c a n t reduct ions i n l eng th compared t o a d u l t - i n f e s t -

i n g nematodes. The width of t h e l a r v a e a t t h e same times

a f t e r h o s t i n f e c t i o n a l s o d i f f e r e d s i g n i f i c a n t l y (Figs . 3 , 4) . Growth p a t t e r n s of nematodes i n nymphal h o s t s were

d i f f e r e n t from those i n a d u l t h o s t s , both i n r a t e of inc rease

i n l e n g t h and width (Figs . 3 , 4) . The s i z e of t h e nematodes

i n nymphal h o s t s increased very s lowly and u s u a l l y only

reached 80% of t h e i r f u l l p a r a s i t i c l e n g t h and width s h o r t l y

before they emerged from t h e h o s t . I n c o n t r a s t , p a r a s i t e s

i n a d u l t h o s t s showed an e i g h t t o nine-fold inc rease i n length

between Days 11 and 17. Inc rease i n maximum width of the

l a r v a e i n a d u l t l o c u s t s occurred i n a s i m i l a r manner, bu t

between Days 8 and 14. I n t h e growth of t h e p a r a s i t e between

nymphal and a d u l t h o s t s , w i th t h e except ion of nematodes de-

veloping i n 1-day-old t h i r d i n s t a r nymphs, t h e longer t h e

#J M. niqrescens l a r v a e spent w i t h i n a h o s t , t h e g r e a t e r was

. 3 , 4 ) .

3 . E f f e c t of t r a n s f e r r i n q M. n iqrescens l a rvae t o a second -

h o s t on the total development t i m e and s i z e of the

p a r a s i t e .

S ingle nematodes t r a n s f e r r e d t o a second h o s t a f t e r

having developed f o r 2 5 days i n t h e f i r s t h o s t increased

s i g n i f i c a n t l y i n mean l e n g t h and remained s i g n i f i c a n t l y longer

i n two h o s t s combined than d id nematodes t h a t had developed

s o l e l y i n one h o s t a able V I I ) . Transfer of t h r e e nematodes

i n t o a second h o s t r e s u l t e d i n a s i g n i f i c a n t i n c r e a s e i n

development time b u t no inc reasc i n s i z e of the l a r v a e .

Transfer of e i t h e r f i v e o r t e n nemae.odes t o a second h o s t

caused no s i g n i f i c a n t d i f f e r e n c e i n e i t h e r development time

o r s i z e of the l a r v a e .

TA

BL

E

VI

I

Th

e e

ffe

ct

of

tra

ns

ferr

ing

~e

rm

is

niq

resc

en

s

larv

ae

to

a

sec

on

d h

ea

lth

y h

os

t (S

ch

isto

ce

rca

qre

qa

ria

) o

n th

e t

ota

l d

ev

elo

pm

en

t ti

me

,, m

ean

len

gth

an

d m

ean

max

imum

w

idth

of

the

pa

ras

ite

.

Mer

mis

n

iqre

sc

en

s

-

Num

ber

of

# o

f In

fec

tiv

e

Dev

elo

pm

ent

Mea

n M

ean

Max

. H

ost

s,

Sex

R

ep

lic

ate

B

urd

en

Tim

e o

f L

arv

ae

L

en

gth

2 S

.E.

Wid

th +

S .

E.

1

of

Ho

st

Ho

sts

Pe

r H

ost

(D

ay

s) 2

S.E

. (mm.

~-

I+

, m

ale

2 ,

male

1 , m

ale

2

, m

ale

1 , f

em

ale

2

, fe

ma

le

1 , f

em

ale

2

, fe

ma

le

1 M

eans

wit

hin

e

ac

h

co

up

let

in

th

e s

ame

colu

mn

fo

llo

we

d

by

th

e s

ame

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DISCUSSION

Although prolonged s t o r a g e lowered M. niqrescens egg

v i a b i l i t y (Table I ) , t h e r e was no i n d i c a t i o n why such eggs

f a i l e d t o produce a h igh percentage of maturing l a r v a e . Pro-

longed s to rage may e f f e c t egg development o r the a b i l i t y of

the l a r v a e t o emerge a t ha tching . C h r i s t i e (1929) s t a t e d t h a t

n e a r l y 50% of g. subniqrescens eggs which had been s to red f o r

1 2 months p r i o r t o i n f e c t i o n passed through t h e a l imentary

t r a c t of M. femur-rubrum unhatched. I t i s not known whether

t h i s occurred i n my experiments s i n c e l o c u s t faeces were not

examined. A l t e r n a t i v e l y , t h e e f f e c t s of s to rage may manifest

i t s e l f behav io ra l ly i n t h e l a r v a ' s i n a b i l i t y t o s u c c e s s f u l l y

f i n d and p e n e t r a t e the g u t w a l l of t h e h o s t .

The experiments on ha tching of 3. niqrescens eggs i n -

d i c a t e t h a t ha tching i s s i t e - s p e c i f i c , b u t do no t i n d i c a t e

whether o r n o t hatching occurs i n response t o a h o s t s t imulus .

Although Crofton (1966) s t a t e s t h a t among f r e e - l i v i n g nematodes

ha tching i s c o n t r o l l e d by a nematode s e c r e t o r y f a c t o r , Rogers

(1960) p o i n t s ou t t h a t many an imal -pa ras i t i c nematodes have

l o s t t h e a b i l i t y t o c o n t r o l the ha tching process . The f a c t

t h a t eggs d id

hatching s i t e P ti.

no t hatch i n g u t s l i g a t e d a n t e r i o r t o the' normal

sugges ts t h a t the h o s t provides the i n i t i a l

hatching s t imulus f o r - M. niqrescens . This may be i n t h e form

of a substance which a c t s t o break down the i n n e r , membranous

covering (Denner, 1968) of t h e egg, t h e o u t e r membrane having

been removed dur ing egg washing p r i o r t o t h e i r s t o r a g e . I f

h o s t s e c r e t i o n only p a r t i a l l y broke down the inner membrane,

l a r v a l s t y l e t a c t i o n o r a c t i o n of a nematode-released sub-

s t ance could complete egg membrane breakdown. Rogers and

Somerville (1957) demonstrated t h a t rwnen f l u i d from the h o s t

t r i g g e r e d p a r a s i t i c t r i c h o s t r o n g y l e l a r v a e of sheep t o pro-

duce exsheathing f l u i d , while Rogers (1960, 1966) showed t h a t

h igh pH and h igh CO concen t ra t ion induced i n f e c t i v e eggs of 2

Ascar is lumbricoides t o produce a ha tching f l u i d t h a t broke

down t h e egg s h e l l . However, n e i t h e r t h e pH nor t h e CO con- 2

c e n t r a t i o n of t h e l o c u s t gu t were measured i n t h i s s tudy. I t

must be remembered t h a t g. n i s rescens eggs i n t h e f r e e - l i v i n g

s t a g e s t i l l have an o u t e r membrane. Therefore, unless t h i s o u t e r

membrane i s removed dur ing t h e e g g ' s passage from t h e l o c u s t

mouth t o t h e midgut, t h e hypothesis must be modified t o account

f o r degradat ion of t h i s membrane. However, s i n c e t h i s ou te r

membrane was e a s i l y removed during egg washing i n t h e l abora to ry ,

the passage of t h e eggs through t h e moist h o s t g u t , p l u s t h e

mechanical a c t i o n s of h o s t d i g e s t i o n , p a r t i c u l a r l y i n t h e . -

- 38 -

i n f e c t e d l o c u s t s .

I t sometimes took up t o 48 hours a f t e r egg i n g e s t i o n

t o f i n d t h e p a r a s i t i c l a r v a e i n t h e h o s t haemolymph. However,

C h r i s t i e (1937) claimed t h a t newly-hatched l a rvae could be

found w i t h i n t h e h o s t haemocoel wi th in 20 t o 30 minutes. A l -

though eggs hatched between l 1/2 and 3 hours a f t e r i n g e s t i o n ,

Denner (1968) was a b l e t o f i n d only 3-day-old l a r v a e i n t h e

h o s t haemocoel. I d id n o t observe i n i t i a t i o n of g u t w a l l

p e n e t r a t i o n , bu t l a rvae were seen emerging from t h e gu t w a l l

t i s s u e i n i n s e c t s a l i n e s o l u t i o n up t o 20 hours a f t e r egg

ha tch . I t i s no t known how long gu t wa l l p e n e t r a t i o n took,

s i n c e l a r v a e were no t observed i n t h e haemolymph u n t i l 48

hours a f t e r egg ha tch , and inL3rmediate observat ions between

24, 48 and 72 hours a f t e r feeding were not made. The only

obse rva t iona l technique employed dur ing these f i r s t 3 days was

t o c e n t r i f u g e decap i t a t ed l o c u s t s and sea rch t h e haemolymph

f o r nematode l a r v a e . Although one t o 3-day-old l a rvae a r e

l e s s than 0.3 mrn long and t h e i r c u t i c l e i s extremely f r a g i l e ,

i t i s inconceivable t h a t c e n t r i f u g a t i o n a t such a low speed

could rup tu re t h e l a r v a l c u t i c l e .

M y r e s u l t s show t h a t t h e p a r a s i t i c phase of M. -

*iqrescens i s s h o r t e r i n d u r a t i o n a t h igher i n f e c t i v e doses. - c h r i s t i e (1929) found a s i m i l a r phenomenon f o r M. - subniqrescens

l a r v a e i n ' h e a v i l y ' i n f e c t e d Romalea microptera and M_. femur-

rubrum, p a r t i c u l a r l y i n female grasshoppers . I n &. niqrescens

i n f e c t i o n s of - F. a u r i c u l a r i a , nematode l a rvae emerged from e a r -

wigs i n f e c t e d wi th 120 eggs i n 11 t o 13 days, from those wi th

t h i r t e e n t o f i f t e e n eggs i n 35 t o 37 days, and i n 55

days when only a s i n g l e p a r a s i t e egg was fed t o t h e h o s t

(Bayl i s , 1944) . Unlike C h r i s t i e (1929) , Baylis found no tend-

ency f o r s h o r t e r p a r a s i t e development time i n female h o s t s .

M y f ind ings support t h e hypothes is t h a t t h e t o t a l

a v a i l a b l e n u t r i e n t s i n t h e h o s t govern t h e l e n g t h of time spent

by t h e p a r a s i t e w i t h i n t h e h o s t ; p a r t i c u l a r l y s i n c e Gordon and

Webster (1971) have shown t h a t 5. niqrescens caused a

s i g n i f i c a n t dep le t ion of haemolymph carbohydrates and f a t body

p r o t e i n s and amino a c i d s i n t h e d e s e r t l o c u s t , e s p e c i a l l y

during t h e l a t e r s t a g e s of i n f e c t i o n . Since they a l s o found

t h a t t h e h o s t does not compensate f o r t h e d r a i n on i t s

n u t r i e n t s by increased food i n t a k e , it fol lows t h a t t h e h igher

the burden of p a r a s i t e s , t he g r e a t e r w i l l be t h e q u a n t i t y of

Emergence from t h e d e s e r t l o c u s t , t h e r e f o r e , probably occurs

when t h e supply of n u t r i e n t s i n t h e .host becomes i n s u f f i c i e n t

t o s a t i s f y the n u t r i t i o n a l requirements of t h e l a r v a e . This

hypothesis ga ins f u r t h e r support from t h e r e s u l t s of t h e t r a n s -

f e r experiments. These r e s u l t s s t r o n g l y support t h e hypothes is

t h a t t h e h o s t in f luences t h e t o t a l p a r a s i t e development t ime.

Once t h e h o s t i s unable t o supply 'an adequate q u a n t i t y of

n u t r i e n t s t o permit f u r t h e r nematode growth, M. niqrescens

emerges from t h e h o s t . Therefore, a change i n t h e con-

c e n t r a t i o n of t h e n u t r i e n t s i n t h e h o s t haemolymph, o r a

change i n t h e haemolymph osmolar i ty a s a r e s u l t of n u t r i e n t

d e p l e t i o n may t r i g g e r th is process .

M. niqrescens completed i t s p a r a s i t i c phase i n t h e

d e s e r t l o c u s t i n a s i g n i f i c a n t l y s h o r t e r time when reared a t

3 5 ' ~ than a t 2 2 Q C . On s e v e r a l occasions it was a l s o noted

t h a t 5. g r e q a r i a nymphs developed a t a slower r a t e a t lower

temperatures , wi th a longer s t a d i a i n each i n s t a r . Many

s t u d i e s (chitwood and Jacobs, 1938 ; Bayl i s , 1947 ; Wigglesworth,

1961; Mahler and Cordes, 1966; Gordon and Webster, 1971, 1972)

i n d i c a t e t h a t t h e r e i s a more r a p i d turnover and u t i l i z a t i o n

of n u t r i e n t s by the h o s t a t h igher r e a r i n g temperatures , hence

r e s u l t i n g i n a g r e a t e r supply of a v a i l a b l e n u t r i e n t s f o r t h e

~ a r a s i t e . Female h o s t s apparen t ly favored - M. n iqrescens l a r v a l

development. The l a r v a e grew and remained w i t h i n female h o s t s

s i g n i f i c a n t l y longer than i n males. Since t h e nematode must

a s s i m i l a t e and s t o r e s u f f i c i e n t n u t r i e n t s dur ing i t s p a r a s i t i c

l i f e t o guarantee success fu l egg product ion dur ing i t s f r e e -

l i v i n g s t a t e i n t h e s o i l (Gordon and Webster, 1971) , a h o s t

t h a t can supply such n u t r i e n t s i n t h e g r e a t e s t q u a n t i t y should

be t h e most s u i t a b l e .

Healthy a d u l t female l o c u s t s have h igher haemolymph

p r o t e i n and carbohydrate concen t ra t ions than males (Gordon

and Webster, 1971) . Kulkarni and Mehrotra (1970) demonstrated

t h a t a d u l t 2. q r e q a r i a females have h igher p r o l i n e , t o t a l

amino a c i d n i t rogen and haemolymph p r o t e i n l e v e l s dur ing c e r -

t a i n per iods of t h e i r a d u l t l i f e than males. Lee (1965) claims

t h a t p a r a s i t i c nematodes may u t i l i z e h o s t carbohydrates a s

s u b s t r a t e s f o r t h e i r own energy metabolism. Host n u t r i e n t s

a l s o provide the energy source requ i red f o r r ap id g. niqrescens

development and p r o t e i n s to rage i n the trophosome (Baylis , 1944; . -

1947) . E i t h e r h o s t haemolymph carbohydrates or s p e c i f i c pro-

t e i n s i n t h e l o c u s t haemolymph may be t h i s c r u c i a l energy

source , s i n c e these a r e s i g n i f i c a n t l y decreased i n a d u l t d e s e r t

l o c u s t s i n f e c t e d wi th M. - niqrescens (Gordon and Webster, 1971;

ord don, Webster and Hislop, pe r sona l communication). Since t h e

+ b iosyn thes i s of aminao a c i d s from carbohydrates and inorganic

n i t rogen has been demonstrated f o r A . - lurnbricoides and Caeno-

r h a b d i t i s b r i q s s a e (Pol lak , 1957; Roths te in and Tomlinson, 1961) ,

a propor t ion of t h e p r o t e i n s r equ i red by M. - niqrescens may o r i -

g i n a t e from h o s t carbohydrates . The f a c t t h a t g. n i s rescens

14 l a rvae can incorpora te l a r g e q u a n t i t i e s of C l euc ine i n t o

p r o t e i n , but no t l a r g e molecules such a s p r o t e i n s , a l s o i n d i -

c a t e s t h a t low molecular weight amino a c i d s i n t h e h o s t haemo-

lymph may form t h e raw m a t e r i a l s f o r both energy product ion and

r a p i d growth. 4. niqrescens l a r v a e inc rease most in . l eng . th be-

tween Days 11 and 2 1 a f t e r egg i n g e s t i o n (Fig. 4 ) , corresponding

c l o s e l y wi th t h e time i n t e r v a l dur ing which t h e l o c u s t

haemolymph carbohydrate l e v e l w a s s i g n i f i c a n t l y lower than

t h a t i n uninfec ted c o n t r o l s (Gordon and Webster, 1971) . Adult

female - S. q r e q a r i a weighed s i g n i f i c a n t l y more than males of

the same age (Gupta and Burr, unpublished o b s e r v a t i o n ) . Thus,

it i s probable t h a t M. n igrescens l a r v a e a t t a i n e d g r e a t e r

l eng ths i n female h o s t s s i n c e they possess a g r e a t e r a v a i l a b l e

r . n u t r i e n t source than male l o c u s t s , a s ind ica ted by t h e i r s i g -

ik. - . n i f i c a n t l y g r e a t e r body weight and the presence of proport ion-

a l l y more a v a i l a b l e haemolymph n u t r i e n t s a s an energy source.

Although t h i s s tudy has shown t h a t h o s t sex (Tables

I V , V) e x e r t s a marked in f luence on nematode development, it

can not e x p l a i n o t h e r r e p o r t s t h a t h o s t sex inf luenced l o c u s t

s u s c e p t i b i l i t y t o mermithid a t t a c k . I t is hard t o envisage

t h a t one sex of - S. q r e q a r i a can avoid i n g e s t i n g mermithid

eggs a s implied by Denner (1968). The same percentage of

M. n iqrescens l a r v a e develop i n e i t h e r sex a t any i n f e c t i v e dose - (Table IV). Since t h e endocrine systems of male and female

i n s e c t s a r e s i m i l a r i n hormonal composition (Novak, 1967;

Highnam, 1967; Highnam and H i l l , 1969; Wigglesworth, 1970) ,

it i s u n l i k e l y t h a t hormonal d i f f e r e n c e s can account f o r t h e

sex-spec i f i c d i f f e r e n c e s i n l o c u s t s u s c e p t i b i l i t y t o mermithid

i n f e c t i o n .

Adult and nymphal 2. q r e q a r i a seem t o c o n s t i t u t e two

d i s t i n c t h o s t c l a s s e s f o r g. niqrescens l a r v a e s i n c e t h e

mean l eng th and mean maximum width growth curves a r e

s i g n i f i c a n t l y d i f f e r e n t (F igs . 3 , 4 ) . The comparative

growth r a t e s of p a r a s i t i c juveni les of g. subniqrescens i n

var ious unspeci f ied grasshopper h o s t s ( i n f e c t i v e dose, one t o

s i x p a r a s i t e s pe r h o s t ) (Denner, 1968) c l o s e l y resemble t h e

growth curve of g. niqrescens i n 2. q r e q a r i a nymphs. However,

my r e s u l t s a r e a l l based on a l a r g e r i n f e c t i v e dose of f i f t y

p a r a s i t e eggs p e r h o s t . Poss ib ly , t h e n u t r i e n t supply i n t h e

grasshoppers which Denner (1968) used was low (due to the

smaller size of his hosts), and nematode growth characteristic

of that of g. niqrescens in adult 2. qreqaria was never

attained.

S E C T I O N I1

Effect of parasite number and host weight on the sex ratio of

Mermis niqrescens in Schistocerca qreqaria.

- 46 -

INTRODUCTION

There have been many sugges t ions a s t o t h e c o n t r o l of

sex r a t i o i n mermithid nematodes. A s t h e number o f g. sub-

niqrescens per grasshopper h o s t increased , t h e p ropor t ion o f

male nematodes increased ( c h r i s t i e , 1929; Denner, 1968) .

c h r i s t i e (1929) concluded t h a t t h e haemolymph environment

was important i n sex de terminat ion . Pe tersen -- e t a l . (1968) and

Petersen and Chapman (1970) concluded s i m i l a r l y f o r Gastro-

m e r m i s sp . and Reesimermis (=Romanomermis) n i e l s e n i T s a i and

Grundmann p a r a s i t e s of mosquitoes. However, Pe tersen and

Chapman (1970) a l s o noted t h a t t h e s p e c i e s o f h o s t inf luenced

R. n i e l s e n i sex r a t i o . Gupta and Burr (unpublished obse rva t ions ) -

demonstrated t h a t an i n c r e a s e i n t h e i n f e c t i v e burden o f

M. niqrescens i n - S. q r e q a r i a caused an i n c r e a s e i n t h e percent -

age of male nematodes and t h a t t h e percentage o f nematodes t h a t

developed i n t o males was independent o f h o s t weight below a

c e r t a i n i n f e c t i v e burden and s t r o n g l y dependent on h o s t weight

above t h a t l e v e l . S t re lkov (1964) noted t h a t t h e sex of F i l i p -

jevimermis s i n q u l a r i s S t re lkov, a p a r a s i t e o f chironomids, was

c o r r e l a t e d wi th h o s t sex i n t h a t male p a r a s i t e s were more o f t e n

found i n male h o s t s and females i n female h o s t s . Frequency of

male Paramermis con to r t a Linslow, a p a r a s i t e of t h e

chironomid, Chironomus c o n t o r t a , increased wi th an inc rease i n

i n f e s t a t i o n l e v e l ( P a r e n t i , 1962a) . A t a given i n f e s t a t i o n

l e v e l , t h e frequency of male nematodes decreased wi th inc reas ing

l eng ths of the Chironomus l a rvae a t t h e time when they were

pene t ra ted by the nematode ( P a r e n t i , 1962b). Pa ren t i (1965)

a l s o found t h a t t h e sex of t h e developing nematode was i n -

f luenced by t h e sex of the p a r a s i t e t h a t f i r s t en te red t h e

h o s t . Once a h o s t had been i n f e c t e d by a nematode of one sex ,

subsequent nematodes t h a t pene t ra ted tended t o d i f f e r e n t i a t e

i n t o t h e oppos i te sex . Pe tersen (1972), however, found t h a t

n e i t h e r t h e s i z e nor the age of t h e mosquito, Culex p ip iens .I

quinquefasc ia tus Say, a f f e c t e d t h e sex r a t i o of t h e 2. n i e l s e n i

l a r v a e . Host d i e t had a no t i ceab le in f luence on t h e sex r a t i o

of t h i s nematode, s i n c e s t a rved and unstarved h o s t s i n f e c t e d

wi th a s i n g l e nematode developed 92% and 13% males, r e s p e c t i v e l y .

The conclusions reached by previous r e sea rcher s a l l

have a common b a s i s i n t h a t they can a l l be c o r r e l a t e d wi th C

t h e t o t a l a v a i l a b l e h o s t n u t r i e n t s . Host age, sex , weight

and spec ies may a l l be i n d i c e s of h o s t n u t r i t i o n a l s t a t e .

LL Depending on t h e t o t a l a v a i l a b l e h o s t n u t r i e n t s and the burden

of p a r a s i t e s p e r h o s t , t he q u a n t i t y of n u t r i e n t s a v a i l a b l e t o

each p a r a s i t e w i l l d i f f e r . Therefore, a s e r i e s of experiments

were done using g. nigrescens i n the d e s e r t l o c u s t t o i n v e s t i -

g a t e t h e e f f e c t of p a r a s i t e burden, h o s t age and h o s t weight

on the sex r a t i o of t h i s nematode.


Ten, 1-day-old a d u l t l o c u s t s of both sexes were each

i n f e c t e d wi th one t o 300 - M. n iqrescens eggs and t e n , 1-day-old

f i f t h i n s t a r nymphs were each i n f e c t e d wi th from f i f t y t o

250 eggs. Immediately a f t e r i n g e s t i n g t h e eggs, t h e l o c u s t s

were weighed and then placed i n i n s e c t a r y r e a r i n g cages.

The h o s t s were k i l l e d 18 days l a t e r and the l a r v a e

from each h o s t were sexed by i d e n t i f i c a t i o n of t h e form of

t h e g e n i t a l primordia. Male nematodes were i d e n t i f i e d a s

e a r l y a s Days 11 t o 14 a f t e r egg i n g e s t i o n , the g e n i t a l p r i -

mordium appearing a s a loose , c e l l u l a r conf igura t ion a t t h e

p o s t e r i o r end of t h e trophosome, which gave t h e l a t t e r s t r u c t u r e

a c h a r a c t e r i s t i c curvature v i s i b l e under the d i s s e c t i n g micro-

scope. These observat ions were c o n s i s t e n t wi th f ind ings of

e a r l i e r work (Bayl i s , 1944, 1947; Gupta and Burr, unpublished

obse rva t ions ) . Male nematodes were v i s i b l y smal ler than

female nematodes of t h e same age.

The percentage s u r v i v a l of 5. niqrescens a t each

i n f e c t i v e burden was c a l c u l a t e d f o r h o s t s of each age and

sex .

RESULTS

I n f e c t i o n o f 1-day-old f i f t h i n s t a r S . q r e q a r i a wi th

120 eggs r e s u l t e d i n 100% male nematodes developing but a s

many a s 180 and 240 eggs had t o be fed t o 1-day-old a d u l t male

and female h o s t s , r e s p e c t i v e l y , t o achieve 100•‹/, male nematodes

developing ( ~ i g . 5 ) . A t each h o s t age, an i n c r e a s e i n t h e

i n f e c t i v e dose of eggs r e s u l t e d i n a h igher percentage of male

nematodes developing.

Mean weights ( four t een r e p l i c a t e s ) of t h e h o s t s a t t h e

t ime of egg i n g e s t i o n were a s fol lows:

1-day-old f i f t h i n s t a r , O.825+O.O4O gms; 1-day-old a d u l t male,

1 .O92 5 . 0 7 1 gms; 1-day-old a d u l t female, 1.47320.162 gms.

~ i f t h i n s t a r nymphs weighed s i g n i f i c a n t l y l e s s (p<O.Ol) than

a d u l t s of e i t h e r sex and a d u l t males weighed s i g n i f i c a n t l y

l e s s (P<0.001) than a d u l t females.

6 The pe rcen t of M_. niqrescens l a r v a e t h a t became males

Figure 5. Effect of infec t ive dose on the sex r a t i o of

Mermis niqrescens i n Schistocerca qreqaria.

Vertical bars represent + 2 standard er rors .

0 40 80 120 160 200 240 280

IN FECTlVE DOSE OF EGGS/HOST

Figure 6 Effect of host weight and infec t ive dose on the

sex r a t i o of Mermis niqrescens i n Schistocerca

s resa r i a . Infect ive dose of paras i tes given for

each l i n e . Vert ical bars represent + 2 standard - er rors .

a=1-DAYOLD 5TH INSTAR HOST

b=l-DAYOLD ADULT MALE HOST

C= I-DAYOLD ADULT FEMALE HOST

.7 .8 .9 1 .O 1.1 1.2 1.3 1.4

MEAN HOST WEIGHT (GMS.) AT EGG INGESTION

M. - niqrescens l a r v a e of 90 and above, and was very weakly

dependent on h o s t weiljht a t i n f e c t i v e doses of - M. niqrescens

l a r v a e of 80 o r l e s s pe r h o s t ( F i g . 6 ) .

For each age and sex of h o s t used, t h e percentage

m o r t a l i t y of g. niqrescens increased with i n c r e a s e i n

i n f e c t i v e dose (F ig . 7 ) . A t an i n f e c t i v e dose of 250

p a r a s i t e eggs pe r h o s t , f o r example, t h e r e was a m o r t a l i t y

of 26%, 34% and 53% f o r 1-day-old f i f t h i n s t a r h o s t s ,

1-day-old a d u l t male h o s t s and 1-day-old a d u l t female h o s t s ,

r e s p e c t i v e l y .

DISCUSSION

I n f e c t i v e dose and h o s t weight a t egg i n g e s t i o n

c l e a r l y inf luenced t h e sex r a t i o o f t h e developing g. niqrescens

l a r v a e i n S . q r e q a r i a . A p o s i t i v e c o r r e l a t i o n between i n f e c t i v e

dose and percentage of male nematodes developing i s i n agree-

ment with previous f ind ings ( C h r i s t i e , 1929; Bayl i s , 1944;

P a r e n t i , 1965; Pe tersen e t a l . , 1968; Pe tersen and Chapman,

1970; Pe tersen , 1972) . However, t h e s i z e of t h e i n f e c t i v e dose

a lone does no t govern t h i s phenomenon. I n f e c t i o n of t h e

earwig, g. a u r i c u l a r i a , with f i v e o r more 3. niqrescens eggs

r e s u l t e d i n lo@/, male nematodes ( ~ a y l i s , 1944) . Pe tersen (1972)

Figure 7 Ef fec t of i n f e c t i v e dose on Mermis n iqrescens

m o r t a l i t y i n Sch i s toce rca q r e q a r i a . V e r t i c a l

ba r s r e p r e s e n t 2 2 s tandard e r r o r s .

1-DAY OLD FIUH INSTAR HOSTS

0 1-DAY-OLD ADULT MALE HOSTS

A 1-DAYOLD ADULT FEMALE HOSTS

100 150 200 250

INFECTIVE DOSE OF EGGS/HOST

quinquefasc ia tus . However, i n M. subniqrescens p a r a s i t i c i n

t 3 . v

R. microptera , i f t h e number of p a r a s i t e s was t h r e e o r l e s s - - 6 2 they were female, when t h e r e were from four t o twenty-three L"+,

t h e r e was a mixture of both sexes , and i n a l l i n s t ances of

twenty-four o r more p a r a s i t e s p e r h o s t a l l t h e developing

larvae were males ( C h r i s t i e , 1929) , I found t h a t h igher I.

i n f e c t i v e doses were requi red t o cause 100% maleness of M_.

niqrescens i n S . q r e g a r i a .

It was pos tu la ted (Sect ion I ) t h a t t h e t o t a l a v a i l a b l e

n u t r i e n t s i n t h e h o s t governed t h e dura t ion of t h e p a r a s i t i c

phase of - M. n iqrescens . A s i m i l a r hypothesis i s now advanced

t o eGPlain t h e development of 100% male nematodes a t h igh i n -

f e c t i v e doses. A l a r g e h o s t , a s i d e n t i f i e d by weight , would

provide more a v a i l a b l e n u t r i e n t s f o r t h e p a r a s i t e than does

a small h o s t . Pe tersen (1972) has shown t h a t when t h e h o s t

is undernourished, male nematodes develop a t lower i n f e c t i v e

burdens. - C . p i p i e n s quinquefascuatus fed a normal d i e t r e -

qu i red seven g. n i e l s e n i p a r a s i t e s per h o s t t o produce a l l

male nematodes, while h o s t s maintained on one-third t h e

normal d i e t throughout t h e p a r a s i t e ' s developmental per iod

requi red only one p a r a s i t e per h o s t t o induce 92% maleness

and t h r e e p a r a s i t e s pe r h o s t t o induce 100% maleness. Hence,

r dose i s increased , the q u a n t i t y of h o s t n u t r i e n t s a v a i l a b l e

t o each p a r a s i t e w i l l be p r o p o r t i o n a l l y decreased, and t h e

r e s u l t w i l l be t h e production of a g r e a t e r percentage of male

nematodes. The h igher m o r t a l i t y of t h e M. niqrescens l a rvae

observed a t l a r g e r i n f e c t i v e doses may be a r e s u l t of com-

p e t i t i o n f o r a v a i l a b l e h o s t n u t r i e n t s . I t i s no t known, however,

a t what time a f t e r egg i n g e s t i o n t h a t p a r a s i t e egg m o r t a l i t y

occurred. I t i s p o s s i b l e t h a t l a r v a l es tabl i shment i n t h e

h o s t haemocoel and/or success fu l l a r v a l p e n e t r a t i o n of t h e

h o s t g u t w a l l was delayed a t h igh i n f e c t i v e doses a s a r e s u l t

of l a r v a l competi t ion f o r space. The e x a c t mechanism i s n o t

knowm

SECTION I11 .

Inhibition of moulting in Schistocerca qreqaria infected by

Mermis niqrescens . \

I n mermithid-parasi t ized i n s e c t s , many changes occur

in processes which a r e normally c o n t r o l l e d i n p a r t by t h e

i n s e c t ' s hormonal balance and/or n u t r i t i o n a l s t a t e . The para-

s i t o i d Stylops s p . can suppress both ovar ian growth and corpora

a l l a t a a c t i v i t y i n the s o l i t a r y bee h o s t , Andrena vaga, pro-

bably by decreas ing h o s t p r o t e i n s y n t h e s i s and p o s s i b l y i n -

d i r e c t l y a f f e c t i n g endocrine a c t i v i t y (Brandenburg, 1956) . Sphaerular ia bombi, a nematode p a r a s i t e of bumble bees,

suppresses h o s t growth and corpora a l l a t a a c t i v i t y , p o s s i b l y

by s e c r e t i n g a t o x i c m a t e r i a l (Palm, 1948) . 4

I

Welch (1960) p o s t u l a t e d t h a t mermithid nematodes i n a

l a r v a l h o s t i n h i b i t h i s t o b l a s t development, e i t h e r d i r e c t l y

by s e c r e t i n g a hormone i n t o t h e h o s t o r i n d i r e c t l y by d i s -

tu rb ing t h e h o s t ' s own hormonal ba lance . U n t i l r e c e n t l y ,

however, l i t t l e information was a v a i l a b l e on t h e n u t r i t i o n a l

r e l a t i o n s h i p between &. niqrescens and t h e d e s e r t l o c u s t , and

of t h e r e s u l t i n g e f f e c t s upon h o s t physiology. Gordon and

Webster (1971) found a r educ t ion i n t o t a l f a t body p r o t e i n

concent ra t ion i n d e s e r t l o c u s t s p a r a s i t i z e d by &. niqrescens .

S i g n i f i c a n t l y , both haemolymph and f a t body p r o t e i n s y n t h e s i s

i n S . q r e q a r i a a r e c l o s e l y implicated i n t h e moulting process

- 58 -

and bo th a r e under neuroendocrine con t ro 1 ( H i

Kearns and Nair (1972) showed t h a t t h e

11, 1962, 1965) .

imaginal moult

of 2. q r e q a r i a was i n h i b i t e d by an i n j e c t i o n of t h e chemoster-

14 i l a n t , t e p a , which caused a decrease i n leucine-C incorpora-

t i o n i n t o the f a t body and subsequent p r o t e i n r e l e a s e . P ro te ins

a r e r equ i red i n t h e epidermal c e l l s j u s t p r i o r t o moulting

(Wigglesworth, 1970) . The i n t e n s e m i t o t i c a c t i v i t y i n t h e

i n s e c t epidermal c e l l s j u s t p r i o r t o moulting r e q u i r e s t h e

a c t i o n of t h e growth hormone, ecdysone, both f o r t h e i n i t i a t i o n

of moulting and t o s t i m u l a t e s u f f i c i e n t q u a n t i t y of f a t body

p r o t e i n s t o be r e l e a s e d i n t o t h e haemolymph f o r con t inua t ion

of t h i s m i t o s j s ,

My o b j e c t i v e s were t o e l u c i d a t e t h e mechanism of i n h i -

b i t i o n of moulting i n 5. s r e q a r i a a s a r e s u l t of g. n i s rescens

p a r a s i t i s m by: (1) measuring ecdysone l e v e l s i n h e a l t h y and

i n f e c t e d l o c u s t s a t t h e time of t h e i r moulting, ( 2 ) determin-

ing haemolymph and f a t body p r o t e i n l e v e l s i n hea l thy and

i n f e c t e d l o c u s t s , and (3 ) measuring t h e incorpora t ion of

leucine-c14 i n t o f a t body p r o t e i n s of hea l thy and i n f e c t e d

l o c u s t s and t h e i r subsequent r e l e a s e i n t o t h e haemolymph.


I n order t o determine whether o r not i n h i b i t i o n of

the imaginal moult i n i n f e c t e d l o c u s t s was due t o a decrease i n

haemolymph ecdysone l e v e l , t h e fol lowing procedures were under-

2 taken . Using a d isposable micropipe t te , 25 of haemolymph

pe r l o c u s t were removed from each h e a l t h y l o c u s t through a

small puncture i n t h e a r t h r o d i a l membrane a t t h e base of t h e

second p a i r of l e g s . This was done 7 days a f t e r they had

moulted i n t o t h e f i f t h i n s t a r and s o corresponded t o t h e 12 t o

2 4 hour per iod p r i o r t o t h e imaginal moult. The ecdysone was

e x t r a c t e d four t imes from t h e haemolymph wi th 3 m l of 65%

methanol i n wa te r - The e x t r a c t was evaporated a t 64-66 '~ , 5

e x t r a c t e d a f u r t h e r t h r e e t imes wi th 10 m l of pure methanol,

and then evaporated t o a f i n a l volume of 1 m l .

Volumes of from 1 t o 2 0 of e x t r a c t from hea l thy

l o c u s t s were i n j e c t e d i n t o t h e p o s t e r i o r end of 5-day-old

Musca domestica l a r v a e which had previous ly been l i g a t u r e d

midway along t h e l o n g i t u d i n a l body a x i s (Fig. 8 ) . The volume of

e x t r a c t t h a t r e s u l t e d l e a s t 50% of t h e g. domestica l a rvae

2 Ecdysone e x t r a c t i o n technique by cour tesy of D r . D . S . King, Zoecon Corporat ion, 975 C a l i f o r n i a Avenue, Palo Al to , C a l i f o r n i a .

Figure 8 . Musca domestica l a r v a e used i n t h e b ioassay of

ecdysone l e v e l s dur ing t h e imaginal moult i n

hea l thy and i n f e c t e d Schis tocerca q r e q a r i a .

A: Five -day-old, l a s t i n s t a r M. - domestica l a r v a e , - i n d i c a t i n g t h e l i g a t u r e midway along t h e

l o n g i t u d i n a l a x i s .

B: M. dornestica, 48 hours a f t e r i n j e c t i o n of

ecdysone e x t r a c t from h e a l t h y l o c u s t s i n t o

p o s t e r i o r end of l a r v a . I n j e c t i o n s i t e

ind ica ted by arrow.

- 6 1 -

developing pupal c h a r a c t e r i s t i c s i n t h e region p o s t e r i o r t o

. . t h e l i g a t u r e wi th in 4 8 hours a f t e r m 3 c c t i o n was used as the

c r i t e r i o n f o r ecdysone l e v e l i n t h e e x t r a c t s .

Haemolymph p r o t e i n measurements were made d a i l y , begin-

ning 2 4 hours a f t e r t h e l o c u s t nymph had moulted i n t o t h e f i f t h

i n s t a r , and te rminat ing when a l l t h e c o n t r o l i n s e c t s had com-

p l e t e d t h e imaginal moult. Using a d i sposab le micropipe t te ,

5p1 samples of haemolymph were obtained from each l o c u s t a s des-

c r ibed i n t h e haemolymph ecdysone l e v e l experiment. Each

l o c u s t was used once, and s i x l o c u s t s were used a t each t ime.

Each haemolymph sample was added t o 2 m l of 5% tr i -

c h l o r o a c e t i c a c i d (TCA) i n o rde r t o p r e c i p i t a t e t h e p r o t e i n .

. . ,,-,L- -- L-L- ---- ----L - 2 L - - 2 ~ L G L A ~ A L R L G w a s w a s l l e u L W L C ~ h i an e i i l a r l u i : ether j 1: ij

mixture, and cen t r i fuged a f t e r each washing. The samples were

then assayed f o r t o t a l haemolymph p r o t e i n according t o t h e

method of Lowry, Rosebrough, Fa r r and Randall (1951) . A c a l i -

b r a t i o n curve was done us ing known concen t ra t ions of bovine

serum albumin (BSA) a s s tandards . The o p t i c a l d e n s i t y of each

sample was read a t 500 mp using a Unicam SP500 spectrophotometer.

For f a t body p r o t e i n a n a l y s i s , approximately 1 mg of lyo-

p h i l i z e d v i s c e r a l f a t body was weighed on a Cahn gram e lec t roba-

l ance and homogenized i n 5 m l of 5% TCA. Af te r c e n t r i f u g a t i o n ,

t h e t o t a l f a t body p r o t e i n was determined a s descr ibed f o r t h e

haemolymph p r o t e i n s .

There was s i g n i f i c a n t l y l e s s p r o t e i n i n t h e f a t body

of i n f e c t e d 7-day-old f i f t h i n s t a r nymphs than i n h e a l t h y ones

of t h e same age. Thus, f a t body from l o c u s t s of t h i s age was

used t o determine t h e e f f e c t s of p a r a s i t i s m on p r o t e i n synthe-

s is . The f a t body t i s s u e was incubated i n a medium con ta in ing

a l l e s s e n t i a l amino a c i d s ( ~ e d i u m A , Stephenson and Wyatt,

14 1962; See Appendix 11) wi th leucine-C ( s p e c i f i c a c t i v i t y

311 m ~ i / r n M ) s u b s t i t u t e d f o r l e u c i n e . Osborne, C a r l i s l e and

E l l i s (1968) noted t h a t t h i s medim was s u i t a b l e f o r incubat ion

of 5. g r e q a r i a f a t body. Measurement of f a t body p r o t e i n

s y n t h e s i s was done according t o Kearns and Nair (1972) wi th

t h e fol lowing except ions . I n e s t i m a t i n g t h e amount of p r o t e i n

i n each sample a f t e r incubat ion (Lowry e t a l . , 1951) , 0.25 m l

14 of e x t r a c t was used. For each sample, the C - a c t i v i t y was

measured over a 10 minute per iod a t a count ing e f f i c i e n c y of

84%, using a Packard Tri-Carb-3003 Liquid S c i n t i l l a t i o n Spectro-

meter. The s p e c i f i c a c t i v i t y of t h e t o t a l p r o t e i n i n each

l o c u s t f a t body, a f t e r c o r r e c t i n g f o r background a c t i v i t y ,

was computed i n counts per minute (CPM) pe r mg of p r o t e i n .

A t t h e end of each incubat ion pe r iod , t h e p r o t e i n s r e -

leased by each f a t body i n t o t h e incubat ion medium were twice

- 63 -

p r e c i p i t a t e d by a d d i t i o n of 2 m l of 5% TCA. The amount of

p r o t e i n and

desc r ibed .

14 i t s C - a c t i v i t y were determined a s p rev ious ly

RESULTS

My r e s u l t s confirm t h a t above a c e r t a i n minimal dose,

and depending on t h e age of t h e h o s t a t t h e time of i n f e c t i o n ,

M. niqrescens i n f e c t i o n r e s u l t s i n an i n h i b i t i o n of 2. g r e q a r i a -

moulting. Table V I I I i n d i c a t e s t h a t f o r each h o s t age group,

t h e r e i s a minimum dose above which t h e next-but-one moult i s

inhibited- This minimum i n f e c t i v e dose i s twenty, t h i r t y and

f o r t y - f i v e nematodes pe r h o s t , f o r 1 -day-old t h i r d i n s t a r ,

1-day-old f o u r t h i n s t a r and 4-day-old f o u r t h i n s t a r nymphs,

r e s p e c t i v e l y . When a 4-day-old f o u r t h i n s t a r nymph i s i n f e c t e d

wi th f o r t y nematodes, it moults i n t o an a d u l t which has de-

formed wings (Fig. 9 ) and f a i l s t o reproduce s u s c e s s f u l l y ,

even i f it escapes dea th by cannibal ism.

The r e s u l t s of the b ioassay f o r ecdysone showed t h a t

t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n ecdysone l e v e l i n i n -

f e c t e d l o c u s t s during t h e time per iod when they should have

moulted i n t o a d u l t s . A volume of 7 . 0 p,1 of ecdysone e x t r a c t

TABLE V I I I

E f f e c t of Mermis n iqrescens p a r a s i t i s m on the moulting of Schis tocerca qreqaria . A t l e a s t s i x t e e n r e p l i c a t e s f o r each h o s t age and i n f e c t i v e dose.

Age of Host Presence of Moult a t time of I n f e c t i v e 3rd t o 4 t h 4 t h t o 5 t h 5 t h t o

I n f e c t i o n Dose I n s t a r I n s t a r Adult

1 -day-old 3rd I n s t a r

1 -day-old 4 t n i n s t a r

4 -day-old 4 t h I n s t a r

1 5 o r Less YES

20 o r More

20 o r Less

30 o r More

35 o r More

YES

YES YES

NO

YES

YES

YES

YES

NO

YES

NO

YES

SOME 1

4-day-old 45 o r More --- YES NO 4 th I n s t a r

Began t h e Imaginal Moult b u t Fa i l ed t o Complete i t .

Figure 9. Mermithid-infected adult Schistocerca qreqari?,

showing deformed wings resulting from parasitism.

from hea l thy , 7-day-old f i f t h i n s t a r 2. qreqar i a was requ i red

t o cause 50.8 - + 1.8% pupal c h a r a c t e r i s t i c s t o appear i n 48

hours i n g. domestica l a r v a e . An i d e n t i c a l volume of ecdysone

e x t r a c t from i n f e c t e d l o c u s t s r e s u l t e d i n 49.0 + 2 .1% of t h e

M. domestica l a rvae d i sp lay ing pupal c h a r a c t e r i s t i c s . -

There was a s i g n i f i c a n t l y lower ( ~ < 0 . 0 0 1 ) haemolymph

p r o t e i n concent ra t ion i n i n f e c t e d than i n h e a l t h y l o c u s t s a t

Days 6 and 7 of t h e f i f t h i n s t a r (Fig. 10) . Since uninfected

l o c u s t s completed t h e i r imaginal moult between Days 6 and 7 1/2

of t h e i n s t a r , t h i s represented a s i g n i f i c a n t l y lower l e v e l

of p r o t e i n s a v a i l a b l e t o t h e l o c u s t dur ing the moulting process .

Although t h e t o t a l f a t body p r o t e i n concen t ra t ion

* i n i n f e c t e d l o c u s t s p a r a l l e l e d t h a t of t h e hea l thy l o c u s t s , i t

d i d s o a t a s i g n i f i c a n t l y lower (P<0.001) l e v e l between Days 6

and 7 of t h e f i f t h i n s t a r (Fig. 1 . This corresponded t o the

same time per iod when t h e haemolymph p r o t e i n concent ra t ion was

s i m i l a r l y decreased (Fig. 10) .

I n both h e a l t h y and i n f e c t e d 7-day-old f i f t h i n s t a r

14 l o c u s t s , i ncorpora t ion of leucine-C i n t o f a t body p r o t e i n s

increased wi th inc rease i n incubat ion t ime. The r a t e of i n -

14 corpora t ion of leucine-C i n t o f a t body p r o t e i n s was, however,

s i g n i f i c a n t l y lower (P<0.001) i n t h e i n f e c t e d l o c u s t s a f t e r

Figure 10. Effect of Mermis niqrescens parasitism on the t o t a l

haemolymph protein leve ls i n f i f t h i n s t a r Schistocerca

qreqaria, infected with f i f t y parasi te eggs 1 day

a f t e r they had moulted i n t o the fourth i n s t a r and

reared a t 3 5 ' ~ . Vert ical bars represent + 2 standard -

er rors .

Figure 11. Effect of Mermis nigrescens parasitism on the

t o t a l f a t body protein leve ls i n f i f t h i n s t a r

Schistocerca qreqaria, infected with f i f t y paras i te

eggs 1 day a f t e r they had moulted i n t o the

fourth i n s t a r and reared a t 3 5 ' ~ . Vertical bars

represent t 2 standard er rors .

HEALTHY T

HEALTHY T

IN FECTJON MOULT

1 2 3 4 5 6 7 8 9

DAYS AFTER ECDYSIS INTO FIFTH INSTAR

incubat ion f o r both 30 am 3 60 minutes (Table I X ) . The q u a n t i t y of p r o t e i n s r e l e a s e d i n t o t h e incubat ion

medium from the f a t body of both h e a l t h y and i n f e c t e d l o c u s t s

increased wi th inc rease i n incubat ion t ime. However, t h e

q u a n t i t y of p r o t e i n s r e l eased from t h e f a t body of i n f e c t e d

l o c u s t s was s i g n i f i c a n t l y lower than t h a t r e l eased from t h e

f a t body of hea l thy l o c u s t s a f t e r 15, 30 and 60 minutes incuba-

t i o n (Table X) . Although the s p e c i f i c a c t i v i t y of t h e p r o t e i n s

r e l eased from t h e f a t body of both h e a l t h y and i n f e c t e d l o c u s t s

increased s i g n i f i c a n t l y (P<0.001) w i t h resp: <:t t o incubat ion

t i m e , t h e r e was no significant difference i n the s p e c i f i c *

a c t i v i t y of t h e r e l e a s e d p r o t e i n s between t h e two groups a f t e r

e i t h e r 15, 30 o r 60 minutes incubat ion i n t h e medium (Table X I ) .

DISCUSSION

The r e s u l t s of t h i s s tudy have shown t h a t t h e r e i s

no decrease i n ecdysone l e v e l a t t h e time of moulting i n i n f e c t e d

l o c u s t s . Consequently, M. niqrescens probably i n h i b i t s moulting

i n t h e d e s e r t l o c u s t by i n h i b i t i n g p r o t e i n s y n t h e s i s and t h e sub-

sequent r e l e a s e of f a t body p r o t e i n s i n t o t h e haemolymph.

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The f ind ing t h a t t h e r e i s no e f f e c t of mermithid p a r a s i t i s m

14 on t h e incorpora t ion of leucine-C i n t o p r o t e i n s r e l eased by

t h e f a t body f u r t h e r i n d i c a t e s t h a t i t is the f a t body, t h e

s i t e of p r o t e i n s y n t h e s i s i n the i n s e c t , t h a t i s a f f e c t e d by

t h e p a r a s i t e .

M y f ind ings have corrobora ted t h e work of Gordon and

Webster (1971), who showed t h a t t h e t o t a l f a t body p r o t e i n

l e v e l of a d u l t female S. g r e q a r i a was s i g n i f i c a n t l y deple ted

by - M . n iqrescens p a r a s i t i s m between Days 16 and 21

a f t e r i n f e c t i o n . I n t h e same s tudy these au thors a l s o noted

t h a t the t o t a l haemolymph p r o t e i n l e v e l s of i n f e c t e d h o s t s d id

not d i f f e r s i g n i f i c a n t l y from t h e l e v e l s i n uninfected h o s t s

throughout t h e i n f e c t i v e pe r iod , a r e s u l t seemingly con t ra -

d i c t o r y t o t h a t found i n t h i s s tudy . I n subsequent experiments

using polyacrylamide d i s c e l e c t r o p h o r e s i s and h o s t s of a d i f f e r e n t

age , however, Gordon, Webster and Hislop (personal communication)

i n d i c a t e d a s i g n i f i c a n t d e p l e t i o n i n haemolymph p r o t e i n eompon-

e n t s a t - B a y 2 1 a f t e r infec t ion . , From thegg f i ~ d i n g s they

concluded t h a t t h e developmental s t a g e of t h e h o s t a t t h e time

of i n f e c t i o n w a s extremely important i n determining t h e e x t e n t

t o which - M . n iqrescens modified h o s t p r o t e i n metabolism. I

a l s o i n d i c a t e d t h a t t h e age of t h e h o s t a t the time of i n f e c t i o n

was important i n t h a t i t s i g n i f i c a n t l y inf luenced t h e dura t ion

r a t e of t h e M. niqrescens l a r v a e (F igs . 3 , 4) ,

Both the t o t a l haemolymph and t o t a l f a t body p r o t e i n

l e v e l s demonstrated f o r uninfected l o c u s t s a r e s i m i l a r t o those

I 1 l e v e l s ind ica ted by previous workers (Kulkarni and Mehrotra, t

1970; Gordon and WeLster, 1971; Kearns and Nair , 1972) . No

comparison can be made, however, s i n c e my r e s u l t s a r e based on

the f i f t h i n s t a r nymph while previous work u t i l i z e d only a d u l t

S . q r e q a r i a . -

There are two means whereby p r o t e i n s y n t h e s i s i n the

f a t body can be i n h i b i t e d . I t i s known t h a t t h e r e i s a neuro-

s e c r e t o r y c o n t r o l of p r o t e i n s y n t h e s i s i n the f a t body of

S . g r e s a r i a (Highnam and H i l l , 1969; Engelmann, 1970) . -

However, s i n c e t h i s s tudy has confirmed t h a t t h e r e i s no e f f e c t

on haemolymph ecdysone l e v e l s of i n f e c t e d l o c u s t s , it i s u n l i k e l y

t h a t f a t body p r o t e i n s y n t h e s i s i s i n h i b i t e d a s a r e s u l t of an

e f f e c t on t h e h o s t ecdysone product ion. Fa t body p r o t e i n

s y n t h e s i s may a l s o be i n h i b i t e d due t o a decrease i n t h e

q u a n t i t y of p r o t e i n precursors i n t h e haemolymph. A s H i l l (1962)

has demon-:trated, the f r e e amino a c i d s i n t h e i n s e c t haemolymph

provide the amino a c i d pool f o r f a t body p r o t e i n s y n t h e s i s .

Gordon and Webster (1971), however, found t h a t a. niqrescens

d id not s i g n i f i c a n t l y a f f e c t the t o t a l amino a c i d l e v e l i n the

- 74 -

haemolymph of in fec ted male and female 2. q r e q a r i a . Although

such a f ind ing would seem t o imply t h a t the i n h i b i t i o n of pro-

t e i n syn thes i s was not due t o a decrease i n the q u a n t i t y of

amino a c i d precursors i n t h e i n f e c t e d h o s t haemolymph, the

evidence may be i n t e r p r e t e d i n s e v e r a l ways when d iscussed i n

r e l a t i o n t o 4. n iqrescens development and assoc ia ted n u t r i -

t i o n a l requirements .

Gordon and Webster (1972) ind ica ted t h a t Days 14 t o 2 1

a f t e r i n f e c t i o n appeared t o be t h e maximal per iod of growth/

p r o t e i n s y n t h e s i s by M. niqrescens l a r v a e . Therefore, i n

a t tempt ing t o f u l f i l l t h e i r h igh requirements f o r d i e t a r y amino

n i t rpgen a t t h i s s t a g e i n t h e i r de~7elopment, the nert?atc?de rzr.1~l.d

e x e r t i t s g r e a t e s t e f f e c t on p r o t e i n turnover i n S . q r e q a r i a .

Since the M_. niqrescens l a rvae used i n my experiments were

between Days 13 and 15 of t h e i r p a r a s i t i c development when they

i n h i b i t e d l o c u s t moulting, t h e i r n u t r i e n t requirements would

be maximal (Gordon and Webster, 1972) . However, s i n c e t o t a l

haemolymph amino a c i d l e v e l s a r e unaffected by g. niqrescens

p a r a s i t i s m (Gordon and Webster, 1971) , I must conclude t h a t t h e

nematode i n d i r e c t l y u t i l i z e s h o s t f a t body p r o t e i n s by inducing

changes i n t h e i r metabolism. 4. niqrescens may s t i m u l a t e pro-

t e i n r e l e a s e from t h e f a t body. Since t h e nematode i s unable

t o a s s i m i l a t e p r o t e i n s and t h e r e i s no evidence of the presence

and Webster, unpublished o b s e r v a t i o n s ) , t hese p r o t e i n s would

e v e n t u a l l y accumulate w i t h i n t h e h o s t haemocoel. Since I

found, however, t h a t M. niqrescens caused a s i g n i f i c a n t de-

1

I p l e t i o n of t o t a l haemolymph p r o t e i n concen t ra t ion i n t h e i

l o c u s t (Fig. 1 0 ) , i t i s u n l i k e l y t h a t t h i s occurs . Decreased

p r o t e i n s y n t h e s i s by t h e i n f e c t e d h o s t f a t body may a l s o be

1

[ due t o a pa ras i t i ca l ly - induced impairment of the f a t body c e l l s

t o incorpora te amino a c i d p recurso r s . This could occur i f t h e

M. n i q r e s c e ~ r - l a rvae sec re ted an i n h i b i t o r y subs tance , A l - - -

though Denner (1968) gave inconclus ive evidence of g.

subniqrescens exudates and s e c r e t i o n s caus ing decreased egg

fecund i ty i n s e v e r a l grasshopper s p e c i e s , I found no evidence

of an i n h i b i t o r y e f f e c t on S. q r e q a r i a a s a r e s u l t of a sub-

s t ance o r i g i n a t i n g from the M. niqrescens l a r v a e (unpublished

observat ion) .

I cons ider it more probable t h a t M_. niqrescens feeds

upon h o s t haemolymph n u t r i e n t s and thus depr ives S . g r e s a r i a

of some of the necessary amino a c i d p recurso r s f o r p r o t e i n

s y n t h e s i s . For t h i s system t o be o p e r a t i v e , however, t h e para-

s i t e would have t o s t i m u l a t e %e l o c u s t f a t body t o c a t a b o l i z e

p r o t e i n s and s o r e l e a s e amino a c i d s i n t o t h e haemolymph; both

f o r t h e nematode's n u t r i t i o n and t o compensate f o r t h e d e p l e t i o n

of haemolymph amino a c i d s r e s u l t i n g from l a r v a l feeding

i n t h e p a r a s i t i z e d - S. q r e q a r i a . This hypothesis has exper i -

mental support i n t h a t it was shown t h a t ca tabo l i smof S .

q r e q a r i a f a t body p r o t e i n s a t 2 weeks a f t e r i n f e c t i o n r e s u l t e d

i n a subsequent d e p l e t i o n of haemolymph p r o t e i n s 1 week

l a t e r (Gordon and Webster, 1971) .

The mechanism(s) whereby M. niqrescens may s t i m u l a t e

p r o t e i n ca tabol i sm ( o r suppress p r o t e i n anabolism) w i t h i n the

f a t body of 5. g r e q a r i a i s no t known. I t can occur as a r e s u l t

of an imbalance i n metabol i te l e v e l between t h e haemolymph

and f a t body o r a s a r e s u l t of an a l t e r a t i o n i n the hormonal

m i l i e u of t h e l o c u s t . While I have shown t h a t t h e r e i s no

d e p l e t i o n i n ecdysone l e v e l s i n i n f e c t e d l o c u s t s during the

moulting process , t h e p o s s i b i l i t y cannot be discounted t h a t

M, niqrescens s e c r e t e s i t s own hormones i n t o the l o c u s t haemo-

lymph. I n v e s t i g a t i o n s by F i she r and Sanborn (1964) and

Rajulu, Kulasekarapandian and Krishnan (1972) have demonstrated

that nematodes can produce substances wi th juveni le hormone

a c t i v i t y and a s t e r o i d hormone, r e s p e c t i v e l y .

My hypothes is on t h e mechanism whereby M. - niqrescens

i n h i b i t s moulting i n the d e s e r t l o c u s t i s a s follows: 5.

niqrescens i n h i b i t s f a t body p r o t e i n s y n t h e s i s i n i n f e c t e d

l o c u s t s by s i g n i f i c a n t l y impair ing t h e incorpora t ion of amino

a c i d precursors from the haemolymph and by concurrent ly

s t i m u l a t i n g catabol ism of f a t body p r o t e i n s ; thus providing

a d i e t a r y source of amino n i t rogen f o r M. niqrescens l a r v a l

growth and development.

- 78 -

Research Topics A r i s i n q o u t o f t h i s P r o j e c t .

Although M. n i q r e s c e n s h a s c o n s i d e r a b l e p o t e n t i a l a s

a b i o l o g i c a l c o n t r o l a g e n t a g a i n s t the d e s e r t l o c u s t , some

a s p e c t s o f t h i s r e l a t i o n s h i p r e q u i r e f u r t h e r c l a r i f i c a t i o n .

1) There i s c u r r e n t l y some con fus ion r e g a r d i n g t h e

taxonomy of g. n i q r e s c e n s and g. subn iq re scens . Although

Cobb (1926) and Denner (1968) m a i n t a i n t h a t M. subn iq re scens

i s a v a l i d s p e c i e s and should b e s e p a r a t e d from g. n i q r e s c e n s ,

N ick l e (1972) h a s synonymized t h e two s p e c i e s . It i s impor t an t

t h a t t h e d i s c r e p a n c y i n t h e taxonomy o f t h e s e s p e c i e s be re-

s o l v e d , s o t h a t t h e r e s u l t s o f my i n v e s t i g a t i o n ~ and t h o s e

a l r e a d y i n t h e l i t e r a t u r e can be used t o best advan tage .

2 ) My- inves t i ga t i ons have i n d i c a t e d t h a t t h e s t i m u l u s

f o r t h e g. n i q r e s c e n s eggs t o h a t c h i n t h e g u t o f t h e d e s e r t

l o c u s t p robab ly o r i g i n a t e s i n t h e h o s t . I t would b e u s e f u l

t o demons t r a t e t h e n a t u r e o f t h i s s t i m u l u s , s i n c e it w i l l be

neces sa ry t o i nduce g. n i q r e s c e n s eggs t o h a t c h as a f i r s t

s t e p i n e s t a b l i s h i n g an & , v i t r o c u l t u r e o f t h e nematode l a r v a e .

3 ) S i n c e t h e p r e s e n t s t u d y shows t h a t t h e burden o f

p a r a s i t e s which a l o c u s t ha rbou r s d i c t a t e s t h e number of male

nematodes t h a t deve lop , it w i l l be n e c e s s a r y t o conf i rm e a r l i e r

i n d i c a t i o n s (Cobb, 1926) t h a t g. n i q r e s c e n s i s p a r t h e n o g e n e t i c

f o r s u c c e s s f u l - i n v i t r o c u l t u r e . I f it i s p a r t h e n o g e n e t i c , t h e n

I only the n u t r i e n t requirements f o r the production of female

nematodes would have t o be determined when i n v e s t i g a t i n g t h e

composition of an in v i t r o c u l t u r e medium. I

i 4) Although previous workers have i n d i c a t e d t h a t

t h e p o s t - p a r a s i t i c s t a g e of g. niqrescens l a s t s f o r 2

o r 3 years , it would be u s e f u l i n a b i o l o g i c a l c o n t r o l pro-

gram t o be a b l e t o shor ten t h i s s o i l - l i v i n g per iod . However,

s i n c e v i r t u a l l y nothing is known of t h e f a c t o r ( s ) t h a t govern

t h e d u r a t i o n of t h e p o s t - p a r a s i t i c phase i n the s o i l , an

avenue of i n v e s t i g a t i o n s i n t h i s a r e a would be b e n e f i c i a l .

5 ) My r e s u l t s show t h a t moulting i s i n h i b i t e d i n

s y n t h e s i s i n t h e f a t body. I t would be u s e f u l t o t e s t m y

hypothesis on t h e mechanism whereby f a t body p r o t e i n s y n t h e s i s

is i n h i b i t e d by g. niqrescens .

6 ) McKinley and Randall (1971) have demonstrated

t h a t t h e r e a r e l a r g e d i f f e r e n c e s i n t h e moisture con ten t

between each of the commonly used l o c u s t and grasshopper

d i e t s . This r e s u l t s i n s i g n i f i c a n t d i f f e r e n c e s i n l o c u s t

s u r v i v a l and body weight and consequently, l a r g e d i f f e r e n c e s

i n t h e q u a n t i t y of n u t r i e n t s pe r u n i t volume of food consumed

by t h e M. niqrescens l a r v a e . Such f a c t o r s a f f e c t nematode

development and warrant f u r t h e r i n v e s t i g a t i o n .

--.

- 80 -

7 ) Although i t was noted t h a t 5. niqrescens l a r v a e

a t t a i n e d a g r e a t e r s i z e under longer r e a r i n g cond i t ions , it

E i s no t known what e f f e c t t h e d u r a t i o n of p a r a s i t i c develop-

I I ment had on e i t h e r the number of eggs l a i d by t h e mature L

nematode females o r on t h e v i a b i l i t y of these eggs. It may be

p o s s i b l e t o remove a p a r a s i t e before it completes i t s para-

s i t i c development and s t i l l have v i a b l e eggs l a i d a t t h e

te rminat ion of p o s t - p a r a s i t i c development.

8) The r e s u l t s of my experiments e l a b o r a t e on t h e

q u a n t i t a t i v e r a t h e r than t h e q u a l i t a t i v e n u t r i e n t r e q u i r e -

ments of M. niqrescens . Future r e s e a r c h should s t r e s s t h e

i n p r t a n c e nf e l i i c ida t ing t h e q u a l i t a t i v e n u t r i e n t r e q u i r e - *

ments a s a necessary f i r s t s t e p t o t h e es tabl i shment of an

i n v i t r o c u l t u r e . -

REFERENCES

Bayl i s , H. A . 1944. Observations on t h e nematode Mermis n iqrescens and r e l a t e d s p e c i e s . Paras i to logy. 36:122-132.

Bayl i s , H. A . 1947. The l a r v a l s t a g e s of t h e nematode Mermis n iqrescens . Paras i to logy. 38: 10-16.

Brandenburg, J. 1956. Das Endokrine System des Kopfes von Andrea vaqa PZ. ( I n s . ~yrnenopt . ) und Wirkung de r S t y l o p i s a t i o n (Stylops, I n s . S t r e p s i p t .) . Z e i t s c h r i f t f u r Morphologie und Oekologie d e r T ie re . 45 : 343 -364.

C h a t t e r j e e , P. N. and P. Singh. 1965. Mermithid p a r a s i t e s and t h e i r r o l e i n n a t u r a l c o n t r o l of i n s e c t p e s t s . Indian F o r e s t e r . 91: 714-721.

Chitwood, B. G. and L. Jacobs. 1938. Stored n u t r i t i v e m a t e r i a l s i n t h e trophosome of t h e nematode, Aqamermis decaudata ( ~ e r m i t h ' i d a e ) . J. Wash. Acad. Sc i . 28 : 1 2 -13.

Christie, 2. I?, 1 9 2 9 . .Some observat ions on sex i n t h e Mermithidae . J. Exp . Zoo1 . 53 : 59-76.

C h r i s t i e , J. R. 1936. Li fe h i s t o r y of Aqamermis decaudata, a nematode p a r a s i t e of grasshoppers and o t h e r i n s e c t s . J. Agric . Res . 52 : 161-198.

C h r i s t i e , J. R. 1937. Mermis subniqrescens , a nematode p a r a s i t e of grasshoppers . J. Agric . Res. 55:353-364.

Cobb, N. A . 1926. The spec ies of M e r m i s : a group of very remarkable nemas i n f e s t i n g i n s e c t s . J. P a r a s i t . 1 2 : 66-72.

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APPENDIX 1

Composition of insect

developmental

Constituent

NaCl

CaC12

NaHCO 3

studies.

saline solution for Mermis nigrescens

APPENDIX 2

Composition of medium A used f o r incubat ing Schis tocerca

g r e q a r i a f a t body.

Const i tuent mq/100 m l

K C 1 2 98

NaH2 PO4

Malic a c i d

L-arginine H C 1 70

L-asparagine 35

L-aspart ic a c i d 3 5

L-cysteine 8

L-cys t i n e 2 - 5

L-glutamic a c i d 60

Const i tuent

L-glutamine

Glycine

L-Histidine H C 1

L-isoleucine

L-Iysine H C 1

L-me th ion ine

L-phenylalanine

L-proline

L-serine

L-threonine

L-tryptophan

L-tyrosine

L-valine

The s o l u t i o n a l s o contained s t reptomycin s u l f a t e (25 pg/ml)

and p e n i c i l l i n

6.5 wi th NaOH.

a c i d s wi th the

G (250 uni ts /ml) , and t h e pH was ad jus ted t o

The CaC12 was added a f t e r mixing t h e amino

o t h e r s a l t s t o avoid p r e c i p i t a t i o n .

- 90 7

CURRICULUM VITAE

Name :

Place and year of B i r th :

Education:

Awards :

Experience:

S t u a r t Melvyn Craig

England, 1947 . Facul ty of Science, Simon Frase r Univers i ty , Burnaby, B. C .

B. Sc. ( ~ i o l o g y ) 1970

Facul ty of Science Simon Frase r Univers i ty ~ u r n a b ~ , B. C.

Graduate S tudies

P res iden t ' s Research Grant 1972

Research A s s i s t a n t , Department of B io log ica l Sciences, Simon Frase r Univers i ty ,

1968 - 1970 (part- t ime)

Teaching A s s i s t a n t , Department of B io log ica l Sciences, Simon Frase r Univers i ty ,

General Manager, F rase r Val ley Mosquito Control

Board Mission Ci ty , B r i t i s h Columbia,

Canada

Publ ica t ions :

Papers given a t the fol lowing meetings :

Webster, J. M, and S. Craig. 1970. The d i r e c t e f f e c t of p l a n t and i n s e c t hormones on Cephalobus sp . J. Nematol . 1: 308.

Eighth Annual Meeting of t h e Socie ty of Nematologists. San Francisco, C a l i f o r n i a . August, 1969.

Northwest Mosquito and Vector Control Associa t ion , Tenth Annual Meeting. V i c t o r i a , B r i t i s h Columbia. October, 1970.

The J o i n t Meeting of t h e Entomol. Soc . Amer . , Entomol . Soc . Queb . , Entomol . Soc. Can. Montreal, Quebec. November, 1972.

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